Visualizing the Replication Cycle of Bunyamwera Orthobunyavirus Expressing Fluorescent Protein-Tagged Gc Glycoprotein

Cells and viruses.BHK-21 and BSR-T7/5 (4) cells were maintained in Glasgow minimal essential medium (GMEM; Invitrogen) supplemented with 10% tryptose phosphate broth, 10% fetal calf serum, and, for BSR-T7/5 cells only, 1 mg of Geneticin (G418) sulfate (Calbiochem) per ml, as described previously (22, 44). A549 cells and Vero E6 (ATCC C1008) cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum. Stocks of wild-type (wt) and recombinant BUNV (rBUNV) were plaque purified in BHK-21 cells. Working stocks were grown by infecting BHK-21 cells in a 175-cm² flask with 500 μl of elite stock and harvesting the supernatant when extensive cytopathic effect (CPE) was apparent.

Titers of the working stocks were determined by plaque assay as previously described (48).

Antibodies.Rabbit antisera against BUNV particles (anti-BUN) and BUNV N protein (anti-N) and mouse monoclonal antibody (MAb) 742 against BUNV Gc protein were described previously (18, 49). MAbs against GM130, a Golgi matrix protein, were purchased from BD Bioscience; MAbs against α-tubulin (CP06) were from Calbiochem, and those against eGFP (ab1218) were from Abcam. Goat anti-rabbit antibody conjugated with fluorescein isothiocyanate (FITC) was purchased from Sigma, and goat anti-mouse antibody conjugated with Cy5 was purchased from Chemicon International Inc.

Plasmids.Plasmids that generate full-length BUNV antigenome RNA transcripts [pT7riboBUNL(+), pT7riboBUNM(+), and pT7riboBUNS(+)] have been described previously (3, 22). pT7riboBUNMGc-eGFP and pT7riboBUNMGc-mCherry were constructed by replacing the coding region for residues 501 to 826 of the glycoprotein precursor in pT7riboBUNM(+) with that of either eGFP (11) or mCherry red protein (36). pT7riboBUNM(+) was first modified to introduce a unique SacI restriction site at nucleotide (nt) 2534 of the cDNA by PCR mutagenesis. The coding regions for eGFP and mCherry were amplified by PCR from plasmids pEGFP-N1 and pmCherry (both Clontech Laboratories), with the introduction of Bsu36I and SacI restriction enzyme sites at the 5′ and 3′ ends. The DNA products were ligated into modified pT7riboBUNM(+) that had been digested with Bsu36I (site at nt 1549 in the BUNV M segment cDNA) and SacI (Fig. 1). All constructs were confirmed by DNA sequence analysis. The primers used and details of PCR are available upon request.

Virus rescue by reverse genetics and virus growth curves.Rescue experiments were performed essentially as described previously (22). Briefly, BSR-T7/5 cells were transfected with a mixture of plasmids comprising 1.0 μg each of pT7riboBUNL(+), pT7riboBUNS(+), and either pT7riboBUNMGc-eGFP or pT7riboBUNMGc-mCherry. At 5 h posttransfection, 4 ml of growth medium was added, and incubation continued for 5 to 11 days at 33°C. The expression of either eGFP- or mCherry-tagged Gc proteins by the transfectant viruses (rBUNGc-eGFP and rBUNGc-mCherry, respectively) was initially examined under a fluorescence microscope, and plaques were isolated on BHK-21 cells. Elite virus stocks were produced by infecting BHK-21 cells in a 25-cm² flask with a single plaque and growing the cultures at 33°C until CPE was evident. To monitor virus growth, BHK-21 cells in 35-mm-diameter petri dishes were infected at a multiplicity of infection (MOI) of 0.1 PFU per cell, and supernatants were harvested at various times after infection. Virus titers were determined by plaque formation on BHK-21 cells.

Metabolic radiolabeling, immunoprecipitation, and Western blotting.Procedures for metabolic radiolabeling and immunoprecipitation of BUNV proteins were described previously (39). Briefly, at 24 h postinfection (p.i.), cells were labeled with [³⁵S]methionine (80 μCi) for 1 h and then lysed, on ice, in 300 μl of nondenaturing radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 1% Triton X-100, 300 mM NaCl, 5 mM EDTA) containing a cocktail of protease inhibitors (Roche). BUNV glycoproteins were immunoprecipitated with anti-BUN serum that had been conjugated to protein A-Sepharose (Sigma) and were analyzed by SDS-PAGE under reducing conditions. For Western blotting, cell lysates of infected BHK-21 cells were separated on 4 to 12% gradient polyacrylamide NuPAGE gels (Invitrogen) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was reacted with appropriate antibodies, and signals were detected by chemiluminescence as described previously (20).

PNGase F digestion.Immunoprecipitates of infected cells were denatured in 30 μl of denaturing buffer (0.5% SDS and 1% β-mercaptoethanol) at 100°C for 10 min and cooled to room temperature. The samples were digested with 4 mU of peptide N-glycosidase F (PNGase F; New England Biolabs) in 40 μl of reaction buffer (50 mM sodium phosphate [pH 7.5], 0.5% SDS, 1% β-mercaptoethanol) for 20 h at 37°C. Treated samples were analyzed by SDS-PAGE.

Immunofluorescence and live-cell imaging of eGFP/mCherry-tagged BUNV infection.To examine the autofluorescence of recombinant virus particles, 5 μl of infectious supernatant, clarified by centrifugation at 3,000 × g for 5 min to remove cellular debris, was mixed with 5 μl of Mowiol mounting medium (pH 8.5) and examined using a Deltavision 3.5 image restoration microscope (Applied Precision). Observations of virus-infected cells by immunofluorescence were as described previously (40). Briefly, infected cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) before they were costained with specific primary antibodies and secondary antibody conjugates. Localization of fluorescently labeled proteins was examined using either the Deltavision 3.5 restoration microscope or a Zeiss LSM confocal microscope as described in the figure legends. For three dimensional (3D) reconstructions of virus-infected cells, a series of images was captured on the Deltavision microscope with Z-sections of 0.2 μm across the cell. Deconvolution and surface rendering for tertiary reconstruction were processed by Huygens Deconvolution software, version 3.4 (Scientific Volume Imaging).

For live-cell imaging, BHK-21 cells grown on chamber slides (μ-Slide with eight wells; ibidi Gmbh, Munich, Germany) and maintained in CO₂-independent medium (Invitrogen), were infected with rBUNGc-eGFP at an MOI of 0.05 PFU/cell. From 12 h postinfection the images were collected at 10-min interval with a Z-section setting of 1 μm (11 images from top to bottom of cell). The Z-stacked images and video clip were processed using Huygens Deconvolution software.

Generation of recombinant BUNV containing Gc tagged with eGFP or mCherry.Recently, we reported a functional analysis of the BUNV Gc protein and observed that the N-terminal half of the Gc ectodomain (residues 477 to 930 of the precursor polyprotein), which forms two structural domains, was not essential for virus replication in cell culture (41). We created four viruses by reverse genetics carrying deletions in Gc ranging from 218 to 450 amino acids, and while all recombinants were attenuated in cell culture, the virus designated rBUNGcΔ7 that lacked amino acids 480 to 826 of the precursor was the least impaired. Hence, we used this deletion as the basis to insert a foreign protein that would be expressed on the surface of recombinant BUNV particles (41). To facilitate cloning of the foreign gene, we introduced a unique SacI restriction enzyme site at nt 2534 in the BUNV M segment cDNA and made use of the naturally occurring Bsu36I site at position 1549. This would allow replacement of amino acids 501 to 826 of the glycoprotein precursor with foreign sequence and would result in a chimeric protein that maintained the authentic 23 N-terminal residues of Gc and should ensure correct proteolytic cleavage between NSm and the chimeric Gc protein. Therefore, we inserted the coding sequence for either eGFP or mCherry fluorescent proteins into pT7riboBUNM(+) (Fig. 1) and recovered two recombinant viruses (designated rBUNGc-eGFP and rBUNGc-mCherry) by reverse genetics (22). In BHK-21 cells, both recombinant viruses formed smaller plaques than those of wt BUNV or the Gc deletion virus rBUNGcΔ7 (Fig. 2 A). The growth characteristics of the viruses were compared in BHK-21 cells infected at a low MOI (0.1 PFU/cell) at 37°C. In accord with their smaller plaque sizes, both recombinant viruses grew more slowly and to lower titers than wt BUNV (Fig. 2B), with rBUNGc-eGFP virus being more attenuated (100-fold lower titer than wt BUNV) than rBUNGc-mCherry (10-fold lower titer than wt BUNV).

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FIG. 2.

Plaque morphology, growth kinetics, and autofluorescence of recombinant viruses. (A) Comparison of the plaque sizes on BHK-21 cells. Cells were fixed at 6 days postinfection with 4% formaldehyde and stained with Giemsa solution. (B) Virus growth curves on BHK-21 cells at 33°C. Cells were infected with wt BUNV virus or the rBUNGc-eGFP (rGc-eGFP) or rBUNGc-mCherry (rGc-mC) recombinant virus at an MOI of 0.1 PFU/cell. Virus was harvested at the indicated times after infection and titrated by plaque formation on BHK-21 cells. Results are shown as the mean of the duplicate titrations. (C) Autofluorescence of recombinant viruses. The supernatant from infected cells containing released virus particles was mixed with an equal volume of Mowiol mounting medium and observed in the Deltavision microscope. Left and middle panels show the autofluorescence of rBUNGc-eGFP (green) and rBUNGc-mCherry (red) viruses, and the right panel shows the image when green and red viruses were mixed. The enlarged inset shows aggregates of green and red viruses. rGcΔ-7, rBUNGcΔ7.

The supernatant of infected cells was clarified by centrifugation to remove cellular debris, and virus particles in the clarified supernatant showed bright autofluorescence under the fluorescence microscope because every copy of the estimated 650 Gc molecules per viron (29) carried a fluorescent protein moiety (Fig. 2C). Variation in size of the fluorescent dots in both the rBUNGc-eGFP (“green virus”; left panel) and rBUNGc-mCherry (“red virus”; middle panel) viruses was noticeable, suggesting that the larger dots may represent aggregated particles. When the red and green viruses were mixed, evidence for aggregation was noted, as indicated by the yellow signal enlarged in the right-hand panel.

Characterization of chimeric Gc proteins.The proteins synthesized by the recombinant viruses were analyzed by immunoprecipitation of radiolabeled cell extracts (Fig. 3 A). The size of Gc in both recombinant viruses was larger than that of the truncated Gc produced by rBUNGcΔ7 (Fig. 3A). In cells infected by rBUNGc-mCherry, the chimeric Gc appeared as a doublet, with the slower-migrating band more dominant than the faster one (Fig. 3A, lanes 8 and 9). In the case of rBUNGc-eGFP, the presence of eGFP sequences in virus-infected cells was confirmed by Western blotting with anti-GFP antibody (Fig. 3B, lane 4).

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FIG. 3.

Characterization of recombinant BUNV expressing chimeric Gc proteins. (A) Comparison of protein profiles of the recombinant viruses rBUNGc-eGFP (rGc-eGFP), rBUNGc-mCherry (rGc-mC), rBUNGcΔ7 (rGcΔ-7), and wt BUNV. BHK-21 cells were infected with wt or mutant viruses at an MOI of 1.0 PFU/cell or were mock infected, and cells were then labeled with [³⁵S]methionine at 24 h postinfection. Viral proteins were precipitated with anti-BUN antibodies and analyzed on 4 to 12% polyacrylamide NuPAGE gels (Invitrogen) under reducing conditions. The positions of viral proteins and protein molecular mass markers are indicated. (B) Western blot analysis to detect eGFP. BHK-21 cells were infected with wt BUNV, rBUNGcΔ7, or rBUNGc-eGFP stock from passage 2 (P2) or passage 10 (P10) or mock infected, as indicated, and cell lysates were fractionated by SDS-PAGE. After transfer to PVDF membrane, the blots were probed with anti-GFP, anti-N, and anti-tubulin antibodies as shown. (C) Glycosylation analysis of viral Gc proteins. Radiolabeled viral proteins were immunoprecipitated with anti-BUN antibodies, and then precipitates were subjected to digestion with PNGase F (F), as indicated, before fractionation on 4 to 12% polyacrylamide NuPAGE gels (Invitrogen) under reducing conditions. The positions of viral proteins are indicated.

To investigate whether the two bands corresponding to the chimeric Gc-mCherry protein arose due to differences in their N-linked glycosylation states, [³⁵S]methionine-labeled viral proteins were immunoprecipitated and subjected to PNGase F digestion. As seen in Fig. 3C, both forms of the Gc protein (lanes 6 and 7) decreased in size, indicating that the second band was not the result of differential N-linked glycosylation. As there is only one N-linked glycosylation site present on the Gc proteins expressed by rBUNGc-eGFP and rBUNGc-mCherry, the shift of size after the deglycosylation is smaller than that of wt BUNV (lanes 2 and 3) that contains two N-glycosylation sites. The genesis of the second Gc band in rBUNGc-mCherry has not been investigated further, but the pattern of bands remains consistent through repeated passages of the virus (see below).

Stability of the eGFP- and mCherry-tagged recombinant viruses.Upon passage of rBUNGc-eGFP in BHK cells, it was noted that some of the plaques produced by the virus no longer fluoresced under UV light. The number of fluorescent plaques in diluted samples of the different virus passages was assessed and showed that the percentage of fluorescent plaques decreased from 100% (12/12 plaques) at passage 5 to 46% (5/11 plaques) at passage 6 and to 0% (0/12 plaques) at passage 7 (data not shown). To investigate this loss of fluorescence, cells were infected with virus from sequential passages, and viral proteins were analyzed by immunoprecipitation and SDS-PAGE. As seen in Fig. 4 A, faster-migrating bands appeared below the Gc-eGFP bands by passages 2 to 4 and increased in intensity over passages 5 and 6. At passage 6, the Gc-eGFP band showed decreased intensity, and at passage 7 no band corresponding to Gc-eGFP was detectable. A single smaller band was evident at passages 7 and 8 and appeared to be a stable species up to passage 9. The loss of eGFP sequences in virus at passage 10 was confirmed by Western blotting, where no reactivity was observed with the anti-GFP antibody (Fig. 3B, lane 5), and by cDNA sequence analysis of the M segment of the mutant viruses (see below).

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FIG. 4.

Stability of fluorescent protein-tagged Gc in recombinant viruses. (A and B) Protein analysis. BHK-21 cells were infected with wt BUNV, rBUNGcΔ7, and stocks of rBUNGc-eGFP (rGc-eGFP) or rBUNGc-mCherry (rGc-mC) from passages 2 to 9 or 10, as indicated, at an MOI of 1.0 PFU/cell and labeled with [³⁵S]methionine at 24 h postinfection. Viral proteins were precipitated with anti-BUN antibodies and analyzed on 4 to 12% polyacrylamide NuPAGE gels (Invitrogen) under reducing conditions. The positions of viral proteins are indicated. (C) Sequence analysis. Alignment of the deduced amino acid sequences of the NSm-Gc region from wt BUNV, rBUNGc-eGFP passage 9, wt MAGV, and MAGV R2. Above is shown a schematic of this region, with amino acid positions indicated.

In contrast to the observation for rBUNGc-eGFP, no loss of fluorescence was observed in cells infected by rBUNGc-mCherry over 10 passages (data not shown). Protein profile analysis revealed that the banding pattern of the doublet Gc protein was constant in cells infected with virus from different passages, and no smaller species were generated (Fig. 4B). Furthermore, the relative intensities of the two bands were constant across all passages. These data indicate that the chimeric Gc-mCherry protein was more stable than the Gc-eGFP protein.

The protein data suggested that rBUNGc-eGFP had deleted the eGFP coding sequence over sequential passages. Therefore, we determined the nucleotide sequence of the M genome segment of recombinant virus at passage 9 by reverse transcription-PCR (RT-PCR), which revealed that not only the complete eGFP coding region was lacking but also the upstream region coding for the remaining 23 residues at the N terminus of Gc (residues 477 to 499 in the precursor) as well as that encoding the C-terminal 135 residues of NSm (residues 342 to 476) (Fig. 4C). It should be noted that the N-terminal 39 amino acid residues of NSm (residues 303 to 341 in the precursor) were retained; these are highly conserved among different orthobunyaviruses and previously were shown to be required for replication of BUNV and the related MAGV (33, 42). Hence, in all subsequent experiments, only early-passage stocks of rBUNGc-eGFP were used, prepared as described in Materials and Methods. As also shown in Fig. 4C, the deletion of the eGFP coding region and part of NSm in the M segment of rBUNGc-eGFP is reminiscent of the internal deletion found in the M segment of the non-temperature-sensitive (ts) revertant R2 virus derived from MAGV (33). Sequence analysis of the M segments of early and late passages of rBUNGc-mCherry revealed no nucleotide differences (data not shown), in accord with the stability of the protein profiles shown in Fig. 4B.

Visualization of BUNV attachment, assembly, budding, and release.The creation of fluorescent protein-tagged viruses enabled us to visualize virus infection at different stages of the life cycle, including attachment, intracellular trafficking, assembly, and budding. To observe the earliest events of virus infection, BHK-21 cells were incubated with rBUNGc-eGFP for different time periods up to 60 min at 37°C, and then the cells were washed with cold PBS and fixed with 4% paraformaldehyde. To observe later replication events, cells were infected for 60 min and washed, and incubation continued at 37°C as indicated in Fig. 5A. After fixation, the infected cells were counterstained with CellMask Deep Red Plasma Membrane Stain (Invitrogen) prior to confocal examination. As shown in Fig. 5 A, virus particles were found attached to the cell surface after a 10-min incubation. Green virus particles were visible both on the cell surface and within the cytoplasm up to 4 h after initial infection though no new viral glycoprotein synthesis was apparent. By 5 h postinfection, the Golgi complex began to show evidence of nascent viral glycoprotein and discrete fluorescent spots could be observed from 6 h postinfection (Fig. 5A, panels 6 h and later). We interpret these spots as representing progeny virus particles in Golgi complex-derived vesicles, in accord with electron microscopy (EM) observations of infected cells that revealed vesicles containing single or very few (three to four) particles (28).

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FIG. 5.

Time course of rBUNGc-eGFP infection. (A) BHK-21 cells were infected with rBUNGc-eGFP at an MOI of 1 PFU/cell and incubated at 37°C for 10 min to 8 h, as indicated, prior to fixation with 4% paraformaldehyde. Cells were counterstained in red with CellMask Deep Red Plasma Membrane Stain (Invitrogen). Representative green virus particles that were either attached to or internalized into the cells at early stages of infection (1 h to 4 h) are marked by arrows though in some cells multiple particles can be seen attached. (B) BSR-T7/5 cells were infected for 8 h with rBUNGc-eGFP at an MOI of 1 PFU/cell, fixed, and costained with anti-tubulin antibody (in red). Enlarged images of selected regions 1, 2, and 3 show progeny virus particles and the virus assembly site as described in the text.

Bunyaviruses assemble and bud at the membranes of the Golgi complex and then must be transported through the cytoplasm for release from cell. Figure 5B shows a BSR-T7/5 cell infected with rBUNGc-eGFP at 8 h postinfection. Enlarged sections 1 and 3 show virus particles trafficking within the cell and being released at the cell surface, and section 2 shows the “factory” in the Golgi complex where viruses bud and assemble. Based on their sizes, which are identical to those of extracellular particles, most of the intracellular virus particles appear to be individual virions rather than aggregates. In enlarged section 3, some virus particles are seen in close association with, or on the tips of, microtubules, perhaps indicating the involvement of microtubules in the intracellular trafficking and release of virions.

Figure 6 shows the Golgi complex in cells infected with wt BUNV or rBUNGc-eGFP. In both cases the Golgi complex was identified by staining with an antibody to the Golgi complex marker GM130, and colocalization of Gc with this organelle is shown by yellow in the merged images (Fig. 6C and G). In Fig. 6C, the Gc protein was stained with MAb 742, a Gc-specific monoclonal antibody (18). In Fig. 6G and the enlarged image in panel H, green virus particles in cells infected with rBUNGc-eGFP virus were shown both in the cytoplasm and at membranes of the Golgi complex (Fig. 6 E to H). However, no individual virus particles could be detected by staining with MAb 742 (enlarged image in Fig. 6D). It is likely that MAb 742 may not recognize Gc proteins that have already been packed icosahedrally into the virion envelope, perhaps due to conformational alteration of the glycoproteins in assembled particles or spatial hindrance for access of the antibody to the epitope, which is conformational and located at or near the first glycosylation site on Gc (residue 424) (38).

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FIG. 6.

Visualization of virus budding at the Golgi complex. BSRT7/5 cells were infected with either wt BUNV (A to D) or rBUNGc-eGFP (E to H) and costained with antibodies to the Golgi marker GM130 (red). BUNV Gc protein in wt BUNV-infected cells was detected with BUNV Gc-specific MAb742. Colocalization between Gc proteins and the Golgi protein are shown in yellow in the merged images (C and G), and enlarged images are shown in panels D and H.

Morphological changes to the Golgi complex in BUNV-infected cells.BUNV infection of African green monkey-derived Vero cells results in early fragmentation of the Golgi complex (34). Using the fluorescent protein-tagged viruses, we investigated Golgi complex disruption in cells derived from other species. Cells were infected at a low MOI in order to allow viewing of infected and uninfected cells in the same microscopic field, and as shown in Fig. 7 A to H, changes to Golgi complex morphology were also observed in human (A549)- and rodent (BHK and BSR-T7/5)-derived cells though to a lower degree than in Vero cells that were included as a control. For these investigations, the Golgi complex was stained with an antibody to GM130, and we reconstructed 3D images of the green or red recombinant virus-infected cells from Z-stack images obtained by Deltavision restoration microscopy using deconvolution and surface rendering software, as described in Materials and Methods. The Golgi structures in uninfected cells (indicated by asterisks in Fig. 7A to H) were relatively compact, whereas in infected cells the Golgi complex, modified by the insertion of Gc protein, appeared rather expanded or dispersed (indicated by solid arrows). These observations suggest that Golgi complex disruption is a ubiquitous effect on cell structure caused by BUNV infection though the extent differs between different cell types. Figure 7I to L show a time course of Golgi complex disruption in BHK-21 cells infected with rBUNGc-eGFP. Changes in morphology were first seen at 6 h postinfection, and Golgi complexes were completely disrupted by 12 h postinfection. This timescale is much later than that seen in Vero cells, where fragmentation is observed before the detection of viral glycoprotein (34).

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FIG. 7.

Morphological changes to the Golgi complex in BUNV-infected cells. A549 (A and B), BSR-T7/5 (C and D), Vero (E and F), and BHK-21 (G and H) cells were infected with either rBUNGc-eGFP (A to F) or rBUNGc-mCherry (G and H). After fixation, cells were stained with anti-GM130 antibodies to detect the Golgi complex, and 3D images were reconstructed from Z-stack images using Huygens deconvolution and surface rendering software. The Golgi complexes in uninfected cells are marked with an asterisk, and those in infected cells are indicated with an arrow. Nuclei were stained in blue with 4′,6′-diamidino-2-phenylindole (DAPI). Panels I to L show the time course of Golgi complex disruption in BSR-T7/5 cells infected with rBUNGc-eGFP. Cells were fixed at 1, 6, 8, and 12 h postinfection, and the Golgi complex was stained in red with antibodies to GM130.

Live-cell imaging of fluorescent protein-tagged BUNV infection.To visualize the viral replication cycle in real time, BHK-21 cells grown in chamber slides were infected with rBUNGc-eGFP and incubated at 33°C in an environmental chamber on the Deltavision restoration microscope. In this situation, which includes using CO₂-independent growth medium, BUNV replication proceeds considerably more slowly than under standard growth conditions (unpublished data). Images were taken at 10-min intervals from 12 h p.i.; Fig. 8 shows only the images taken at hourly intervals (for the complete video clip, see Video S1 in the supplemental material). Fluorescent virus particles were seen trafficking from the assembly site in the Golgi region to the cell surface up to 21 h p.i. The dynamic live-cell imaging also revealed the morphological change in the Golgi complex from a tightly packed structure to a loose and fragmented structure from 13 h to 16 h p.i. (based on the colocalization with the fluorescent Gc protein). Also noteworthy is the formation of a dense fluorescent mass which we think is most likely accumulation of misfolded glycoproteins in the Golgi region from 17 h p.i. BUNV replication induces apoptosis at late stages of infection (15), and this is visualized as the collapse of the infected cell between 21 and 22 h p.i.

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FIG. 8.

Live-cell imaging of BHK-21 cells infected with rBUNGc-eGFP. BHK-21 cells grown on microscope chamber slides were infected with rBUNGc-eGFP at an MOI of 1 PFU/cell and incubated at 33°C in an environmental chamber mounted on the Deltavision restoration microscope. Images were collected at 10-min intervals from 12 h postinfection with a Z-section setting of 1 μm (11 images from top to bottom of the cell). Images taken at each hour are shown in the figure (for the complete video clip, see Video S1 in the supplemental material).