Almost all of the constituents of the coronavirus RTCs are encoded by the large replicase gene that is comprised of two partly overlapping open reading frames (ORFs), ORF1a and ORF1b. Translation of these ORFs results in two very large polyproteins, pp1a and pp1ab, the latter of which is produced by translational readthrough via a −1 ribosomal frameshift induced by a “slippery” sequence and a pseudoknot structure at the end of ORF1a (
46,
69). pp1a and pp1ab are extensively processed into an elaborate set of nonstructural proteins (nsps) via co- and posttranslational cleavages by the viral papain-like proteinase(s) (PLpro) residing in nsp3 and the 3C-like main proteinase (Mpro) in nsp5 (
17,
51,
64,
66,
77). The functional domains present in the replicase polyproteins are conserved among all coronaviruses (
77). The ORF1a-encoded nsps (nsp1 to nsp11) contain, among others, the viral proteinases (
17,
51,
64,
66,
77), the membrane-anchoring domains (
34,
48,
49), anti-host immune activities (
8,
32,
47,
78), and predicted and identified RNA-binding and RNA-modifying activities (
20,
27,
31,
43,
67,
76). ORF1b (nsp12 to nsp16) encodes the key enzymes directly involved in RNA replication and transcription, such as the RNA-dependent RNA polymerase (RdRp) and the helicase (
2,
7,
11,
18,
29,
30,
33,
45,
60). The nsps collectively form the RTCs; however, the size and complexity of these complexes are unknown.
Coronavirus replicative structures consist of double-membrane vesicles (DMVs) in which the RTCs are anchored (
3,
23,
65). Although hardly anything is known about the mechanism by which the DMVs are induced, recent studies by us and others indicate that the DMVs are most likely derived from the endoplasmic reticulum (ER). Electron microscopy (EM) analyses of infected cells showed the partial colocalization of nsps with an ER protein marker while the DMVs were often found in close proximity to the ER and, occasionally, in continuous association with it (
35,
65). More recently, the DMVs were reported to be integrated into a reticulovesicular network of modified ER membranes, also referred to as convoluted membranes (CMs) (
35). In addition, when expressed in the absence of a coronavirus infection, nsp3, nsp4, and nsp6 were inserted into the ER (
26,
34,
48,
49). When expressed in coronavirus-infected cells, nsp4 appeared to exit the ER and to be recruited to the RTCs (
49). Furthermore, coronavirus replication was severely affected when the formation of COPI- and COPII-coated vesicles in the early secretory pathway was inhibited by the addition of drugs, by the expression of dominant negative mutants, or by depletion of host proteins using RNA interference (
49,
72).
The mechanisms underlying the assembly of membrane-associated replication complexes in cells infected with plus-strand RNA viruses are just beginning to be unraveled. Previous studies have provided valuable information on the formation of the virus-induced replicative structures, resulting, however, in a static view of these processes inherent to the cell biological techniques used. Thus, insight into the dynamics of these structures is largely lacking, certainly in the case of coronaviruses. In the present study, we made the first step to fill this gap by performing live-cell imaging analyses of mouse hepatitis coronavirus (MHV) replicative structures in combination with fluorescent recovery after photobleaching (FRAP) studies. This approach allowed us to monitor the coronavirus DMV-anchored RTCs in real time and generated new insights into the dynamics of these virus-induced structures, revealing striking similarities between the replicative structures induced by MHV and those generated by the unrelated hepatitis C virus (HCV).
MATERIALS AND METHODS
Cells, viruses, and antibodies.
HeLa-CEACAM1a (
75),
Felis catus whole fetus (FCWF) cells (American Type Culture Collection) and murine LR7 fibroblast cells (
36) were maintained as monolayer cultures in Dulbecco's modified Eagle's medium (DMEM−/−; Cambrex BioScience) containing 10% fetal calf serum (FCS; Bodinco BV), 100 IU/ml of penicillin, and 100 μg/ml of streptomycin (both from Life Technologies; this medium is referred to as DMEM+/+).
MHV strain A59, recombinant wild-type MHV (MHV-WT) (
13), recombinant MHV-ERLM (
12), which expresses the
Renilla luciferase (RL) gene, and the recombinant viruses generated in this study, MHV-nsp2GFP (where GFP is green fluorescent protein), MHV-nsp2mCherry, and MHV-nsp2RL, were propagated in LR7 cells.
Antibody directed against double-stranded RNA (dsRNA) (K1) or the GFP was purchased from English and Scientific Consulting Bt. (
58) and Immunology Consultants Laboratory, Inc., respectively. The polyclonal anti-p22 antibody, which is directed against MHV nsp8 (
39), the monoclonal M
N antibody, recognizing the N-terminal domain of the MHV membrane (M) protein (
68), and the polyclonal anti-D3 (nsp2/nsp3) and anti-D11 (nsp4) rabbit antibodies (
9) were kindly provided by Mark Denison, John Flemming, and Susan Baker, respectively. The peptide serum recognizing the C-terminal tail (anti-M
C) of the MHV M protein has been described before (
38).
Plasmids.
The MHV A59 nsp2 gene fragment was generated by reverse transcriptase (RT)-PCR amplification of viral genomic RNA using the primers indicated in Table
1. The obtained PCR product was cloned into the pGEM-T Easy vector (Promega), which resulted in the pGEM-nsp2 plasmid. This plasmid was used as the starting point for the generation of the other nsp2-encoding plasmids that were subsequently used for the generation of recombinant viruses and for expression studies. Gene fragments, encoding C- and/or N-terminal nsp2 deletion mutants, were generated by PCR using the primers indicated in Table
1 and cloned into the pGEM-T Easy vector, generating pGEM-nsp2AB (nsp2 residues 1 to 247), pGEM-nsp2BC (nsp2 residues 122 to 459), and pGEM-nsp2CD (nsp2 residues 247 to 585). The nsp2-encoding gene fragments were subsequently cloned into the pEGFP-N3 vector (Clontech), resulting in pEGFP-nsp2 (where EGFP is enhanced GFP), pEGFP-nsp2AB, pEGFP-nsp2BC, and pEGFP-nsp2CD (Fig.
1B).
Three RNA transcription vectors (pMH54-nsp2EGFP, pMH54-nsp2mCherry, and pMH54-nsp2RL) were generated in order to create recombinant MHVs expressing the gene encoding nsp2 tagged with either EGFP (Clontech), mCherry (Clontech), or
Renilla luciferase (Invitrogen) at the genomic position of the hemagglutinin esterase (HE) gene. These vectors were constructed similarly as described previously for pMH54-nsp4EGFP (
49), with the exception that the nsp2 rather than the nsp4 gene fragment was cloned in frame with either EGFP-, mCherry-, or RL-encoding sequences.
The expression plasmid encoding alpha-tubulin as a yellow fluorescent protein (YFP) fusion construct (pYFP-alpha-tubulin) was obtained from Euroscarf (
1). The pER-GFP construct encoding an ER-retained GFP protein was kindly provided by Frank van Kuppeveld. The GFP coding region in this plasmid was replaced by that of firefly luciferase (Fluc) using conventional cloning techniques, resulting in pER-Fluc. All constructs were confirmed by restriction and/or sequence analysis.
Targeted recombination.
Incorporation of the nsp2 expression cassettes into the MHV genome by targeted RNA recombination, resulting in recombinant MHV-nsp2GFP, MHV-nsp2mCherry, and MHV-nsp2RL viruses, was carried out as previously described (
36). Briefly, donor RNA transcribed from the linearized transcription vectors was electroporated into FCWF cells that had been infected earlier with the interspecies chimeric coronavirus fMHV (an MHV derivative in which the spike ectodomain is of feline coronavirus origin) (
36). These cells were plated onto a monolayer of murine LR7 cells. After 24 h of incubation at 37°C, progeny viruses released into the culture medium were harvested and plaque purified twice on LR7 cells before a passage 1 stock was grown. After confirmation of the recombinant genotypes, passage 2 stocks were grown that were subsequently used in the experiments.
Infection and transfection.
Subconfluent monolayers of LR7 cells were transfected by overlaying the cells with a mixture of 0.5 ml of OptiMem (Invitrogen), 1 μl of Lipofectamine 2000 (Invitrogen), and 1 μg of each selected construct, followed by incubation at 37°C. Three hours after transfection, the medium was replaced by DMEM+/+. Where indicated, 24 h after transfection the cells were inoculated with (recombinant) MHV A59 at a multiplicity of infection (MOI) of 1 to 10 for 1 h before the inoculum was replaced by fresh DMEM+/+.
One-step growth curve(s).
LR7 cells grown in 0.33-cm2 tissue culture dishes were infected in parallel using an MOI of 10 for 1 h at 37°C in 5% CO2. After adsorption, the cells were washed with phosphate-buffered saline (PBS) supplemented with 50 mM Ca2+ and 50 mM Mg2+ three times, and incubation was continued in DMEM+/+. Viral infectivity in culture medium at different times postinfection (p.i.) was determined by a quantal assay on LR7 cells, and the 50% tissue culture infective dose (TCID50) values were calculated.
Metabolic labeling and immunoprecipitation.
Subconfluent monolayers of LR7 cells in 10-cm
2 tissue culture dishes were infected with the viruses indicated in Fig.
2F for 1 h at an MOI of 10, after which the inoculum was removed; the cells were then washed three times with DMEM+/+, and incubation was continued in DMEM+/+. At 5.5 h p.i., the cells were starved for 30 min in cysteine- and methionine-free modified Eagle's medium containing 10 mM HEPES (pH 7.2) and 5% dialyzed FCS. This medium was replaced with 1 ml of a similar medium containing 100 μCi of
35S
in vitro cell labeling mixture (Amersham), after which the cells were further incubated for 3 h. The cells were washed once with PBS supplemented with 50 mM Ca
2+ and 50 mM Mg
2+ and then lysed on ice in 1 ml of lysis buffer (0.5 mM Tris [pH 7.3], 1 mM EDTA, 0.1 M NaCl, 1% Triton X-100). The lysates were cleared by centrifugation for 5 min at 15,000 rpm and 4°C and used in radioimmunoprecipitation studies. Aliquots of the cell lysates were diluted in 1 ml of detergent buffer (50 mM Tris [pH 8.0], 62.5 mM EDTA, 1% NP-40, 0.4% sodium deoxycholate, 0.1% SDS) containing antibodies (4 μl of rabbit anti-nsp2/nsp3). After overnight incubation at 4°C, the immune complexes were adsorbed to Pansorbin cells (Calbiochem) for 60 min at 4°C and subsequently collected by centrifugation. The pellets were washed three times by resuspension and centrifugation with radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 0.1% SDS, 1% NP-40, 1% sodium deoxycholate). The final pellets were suspended in Laemmli sample buffer (LSB) and heated at 95°C for 1 min before analysis by SDS-polyacrylamide gel electrophoresis (PAGE) with 12.5% polyacrylamide gels. The radioactivity in protein bands was quantitated in dried gels using a PhosphorImager (Molecular Dynamics).
Quantitative RT-PCR.
Total RNA was isolated from infected cells using TRIzol reagent (Invitrogen), after which it was purified using an RNeasy minikit (Qiagen), both according to the manufacturer's instructions. Relative gene expression levels of viral (sub)genomic RNA was determined by performing quantitative RT-PCR using Assay-On-Demand reagents (PE Applied Biosystems) as described previously (
14,
52). Reactions were performed using an ABI Prism 7000 sequence detection system. The comparative threshold cycle (
CT) method was used to determine the fold change for each individual gene.
Immunofluorescence confocal microscopy.
LR7 cells grown on glass coverslips were fixed at the times indicated in the text and figure legends after transfection or infection using a 4% paraformaldehyde (PFA) solution in PBS for 30 min at room temperature. The fixed cells were washed with PBS and permeabilized using either 0.1% Triton X-100 for 10 min at room temperature or 0.5 μg/ml digitonin (diluted in 0.3 M sucrose, 25 mM MgCl2+, 0.1 M KCl, 1 mM EDTA, 10 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)], pH 6.8) for 5 min at 4°C. Next, the permeabilized cells were washed with PBS and incubated for 15 min in blocking buffer (PBS-10% normal goat serum), followed by a 60-min incubation with antibodies directed against either nsp4, nsp8, MHV M, EGFP, or dsRNA. After three washes with PBS, the cells were incubated for 45 min with Cy3-conjugated donkey anti-rabbit immunoglobulin G antibodies (Jackson Laboratories), fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G antibodies (ICN), or Cy3-conjugated donkey anti-mouse immunoglobulin G antibodies (Jackson Laboratories). Where indicated, nuclei of cells were stained with TOPRO 3 iodide (Molecular Probes). After four washes with PBS, the samples were mounted on glass slides in FluorSave (Calbiochem). The samples were examined with a confocal fluorescence microscope (Leica TCS SP), or fluorescence intensities were quantified using a DeltaVision RT microscope and software from Applied Precision, Inc. (API).
Time-lapse live-cell imaging and photobleaching.
Subconfluent monolayers of LR7 cells were grown in 0.8-cm
2 Lab-Tek Chambered Coverglasses (Thermo Fisher Scientific and Nunc GmbH & Co. KG). The cells were transfected and infected as described above. Where indicated in the text and figure legends, cells were incubated with or without 1 μM nocodazole in DMEM +/+ at 4°C for 1 h, after which the cells were transferred to 37°C, and incubation was continued. Live-cell digital images of cells, placed in an environmental chamber at 37°C, were acquired at ×100 magnification by the DeltaVision RT microscope from Applied Precision, Inc. (API). Images were deconvolved and analyzed using SoftWorx software (API). Time-lapse movies in QuickTime format were generated using ImageJ software, version 1.41(W. S. Rasband, NIH, Bethesda, MD [
http://rsb.info.nih.gov/ij/ ]). Particle tracking was performed using the MTrackJ plug-in for ImageJ developed by Erik Meijering at the Biomedical Imaging Group Rotterdam (Erasmus Medical Centre, Rotterdam, The Netherlands).
FRAP experiments were performed using the quantifiable laser module (QLM) of the DeltaVision RT microscope at 37°C. For each FRAP experiment, five prebleach images were collected, followed by a 1-s, 488-nm laser pulse with a radius of 0.500 μm to bleach the regions of interest (ROI). In a time frame of 60 s, 52 additional images were captured. Quantitative analysis of the FRAP data was performed using SoftWorx software.
Differential ultracentrifugation and protease protection assay.
Subconfluent monolayers of LR7 cells were transfected and/or infected as described above and washed once with PBS before being scraped in homogenization buffer (HB; 50 mM Tris-HCl [pH 7.2] and 10 mM sucrose) and centrifuged for 5 min at 1,200 rpm. Cells were subsequently resuspended in HB, and homogenized cell lysates were prepared by repeated passage through a 21-gauge needle. The differential ultracentrifugation was performed in a Beckman Coulter Optima Max-E ultracentrifuge using a TLA-55 rotor. First, the homogenized cells were centrifuged for 10 min at 3,000 rpm to remove the nuclei and the cellular debris. The resulting supernatant was next centrifuged for 20 min at 23,000 rpm to separate the intracellular membranes (pellet) from the cytosol (supernatant). Where indicated in the text and figure legends, the intracellular membrane fractions were mock treated or treated with 20 μg/ml proteinase K for 10 min at 20°C in the presence or absence of 0.05% TX-100 before proteinase K was inactivated by the addition of 2 mM phenylmethylsulfonyl fluoride (PMSF). Renilla and firefly luciferase activity in the different fractions was determined using a Dual-Luciferase Assay Kit (Promega) according to the manufacturer's instructions.
EM procedures.
HeLa-CEACAM1a cells infected with recombinant MHV-nsp2GFP were processed for conventional EM and cryo-immuno-EM (IEM) at 8 h p.i. as previously described (
63,
72). Cryo-sections were immunolabeled using a polyclonal anti-GFP (Abcam, Cambridge, United Kingdom) antibody, followed by incubation with protein A-gold conjugates prepared following an established protocol (
63). Sections were viewed in a JEOL 1010 or a JEOL 1200 electron microscope (JEOL, Tokyo, Japan), and images were recorded on Kodak 4489 sheet films (Kodak, Rochester, NY).
DISCUSSION
In this study, the dynamics of the coronavirus replicative structures was analyzed for the first time by performing live-cell imaging of coronavirus-infected cells in combination with FRAP analyses. Ideally, one may prefer to visualize these structures by using recombinant viruses expressing tagged versions of an nsp in the context of the replicase precursor proteins; however, such recombinants are currently not available. Therefore, we applied an alternative approach in which the replicative structures were visualized by the expression of fluorescently marked nsp2 proteins in
trans in MHV-infected cells. Although this protein is dispensable in virus replication and the formation of the viral RTCs (
24), this protein was more efficiently recruited to the RTCs than other nsps, at least when expressed in
trans (data no shown). The tagged nsp2 proteins were found by immunofluorescence analyses to colocalize with several RTC markers, such as nsp8, nsp4, and dsRNA. nsp8 was recently shown to contain RdRp activity and has been proposed to function as a primase (
27) while nsp4 has a critical role in directing coronavirus RTC/DMV assembly (
9). dsRNA molecules, which are readily detected in coronavirus-infected cells (
49), are likely to represent replicative intermediates. Consistently, nsp2-GFP was shown by immuno-EM analysis to be efficiently recruited to the virus-induced DMVs and CMs in MHV-nsp2GFP-infected cells. Previously, newly synthesized viral RNA as well as (viral) dsRNA had been found to be associated with the DMVs (
23,
35,
65) while all nsps studied to date have been localized to the DMVs and CMs (
16,
24,
49,
51,
61). Taking all these observations together, we conclude that the expressed nsp2 fusion proteins are recruited to the coronavirus RTCs, which are anchored to DMVs. Importantly, this localization corresponds with the previously reported distribution of nsp2 (
5,
21,
24,
62).
The nsp2 fusion proteins were associated with the cytoplasmic face of the DMVs/CMs. After selective permeabilization of the plasma membrane, antibodies directed against the GFP tag were able to detect the nsp2 fusion protein. Furthermore, the large majority of the membrane-associated nsp2 was sensitive to protease treatment in the absence of detergents. Apparently, no appreciable fraction of nsp2 was protected by the membranes, which indicates that this protein is not targeted into the lumen of the DMVs. In agreement with the association of nsp2 to the DMV external surface, nsp2-GFP could also be detected in the sections prepared for immuno-EM, in which the DMVs appeared as empty vesicles that lacked the inner membrane. Recently, van Hemert and coworkers (
70) demonstrated that dsRNA, nsp5, and nsp8 present in partially purified severe acute respiratory syndrome (SARS)-CoV RTC preparations were protected by membranes from nuclease or protease treatment. Interestingly, however, this was not the case for the very large nsp3. Thus, it appears that some nsps (e.g., nsp5 and nsp8) are protected by membranes, e.g., by their localization inside the DMVs, while others are not (e.g., nsp2 and nsp3). This raises intriguing questions about the overall structure of the coronavirus RTCs and their association with cellular membranes.
As nsp2-GFP was recruited both to single DMVs and to DMV/CM assemblies but not to any other cytoplasmic structure, we conclude that the small, mobile nsp2-positive structures are likely to correspond to single DMVs while the large immobile nsp2-positive structures probably represent the DMV/CM assemblies. Correlative light-electron microscopy, in which live-cell imaging of fluorescently tagged proteins together with immunogold labeling of ultrathin cryosections of the same cells is combined (
71), will be required to unequivocally prove this point. As our results indicate that single DMVs are mobile but that the DMV/CM assemblies are not, one might speculate that newly formed DMVs are able to freely move around until they are “captured” by the DMV/CM assemblies.
The small nsp2-positive foci, supposedly corresponding to single DMVs, traffic through the cell in a microtubule-dependent fashion. Several lines of evidence support this conclusion. The fluorescent structures appeared to traffic on specific cellular tracks, displaying velocities and saltatory movements typical for microtubule-mediated transport (
40), while such movements were not observed in the presence of nocodazole. Furthermore, when cells lacking a microtubular network were infected with MHV, the nsp2-positive structures did not accumulate in the perinuclear region of the cell but, rather, were scattered throughout the cytoplasm. Disruption of microtubule-mediated transport of DMVs, however, had no significant impact on coronavirus RNA replication. Trafficking of viral replication complexes along microtubule tracks has previously also been observed for other plus-strand RNA viruses, e.g., HCV (
74), poliovirus (
10,
19), and the double-stranded DNA vaccinia virus (
57). Strikingly, also for these viruses replication was not affected or only modestly affected by the disruption of microtubules (
6,
19,
59).
While the trafficking of viral replication complexes along microtubule tracks has been documented for several viruses, the dynamics of these structures has so far been reported in detail only for HCV (
74) and vaccinia virus (
59). Live-cell imaging of vaccinia virus RTCs demonstrated that only the small (early), and not the large (late), replication sites displayed microtubule motor-mediated motility (
59). In the case of HCV, Wölk and coworkers used replicons harboring a GFP insertion in NS5A. Again, two distinct patterns of NS5A-GFP fluorescence were reported: (i) large structures which showed restricted motility and (ii) small structures which showed fast, saltatory movements over large distances. Interestingly, the NS5A-GFP-positive structures displayed a static internal architecture without detectable exchange of NS5A within or in between these structures, as determined by FRAP analyses. Although the experimental approach of this study differs from ours (i.e., the HCV NS5A is an essential replicase protein and was expressed in the context of the viral polyprotein), the dynamics of the HCV replicative structures show several remarkable similarities with those of MHV.
The large MHV DMV/CM assemblies very likely correspond to the recently reported reticulovesicular network of modified ER membranes that is connected to clusters of interconnected DMVs found in SARS-CoV-infected cells (
35). From this perspective, it is not surprising that these large assemblies of interconnected ER and DMVs are not able to traffic on microtubule tracks. Interestingly and similar to our observations, movement in HCV and vaccinia virus was observed for only the small RTC assemblies and not the large ones (
59,
74). The fast saltatory movements of the small HCV fluorescent foci were shown to occur independently of ER dynamics (
74). Whether this also holds true for MHV remains to be established.
Despite the movement of the small nsp2-positive foci, the coronavirus replicative structures turn out to be inherently static entities. No recovery of fluorescence was observed when (part of) the nsp2-positive structures were photobleached. Apparently, the nsp2 protein, once recruited to the RTCs, is not exchanged by nsp2 protein occurring in the cytoplasm or at other DMVs. This result was confirmed by the observation that preexisting RTCs did not exchange fluorescence after fusion of cells expressing either a green or a red fluorescent nsp2. Our data thus indicate that recruitment of nsp2 occurs only during RTC assembly. Again, similar results were obtained for the HCV RTCs, which also displayed a lack of fluorescence recovery after photobleaching (
74). We hypothesize that during RTC assembly, the coronavirus nsp2 is captured within an elaborate network of protein-protein interactions. Indeed, for the nsp2 of SARS-CoV, a large number of viral protein interaction partners have been identified including nsp2, nsp3, nsp6, nsp8, and nsp11 (
50,
73). Other coronavirus nsps also appear to be contained in rigid protein-protein interaction networks (
28,
50,
73; also unpublished results).
Our findings have important consequences for our understanding of RTC assembly and functioning. The lack of exchange of nsps present in different RTCs fits well with the model of RTC maturation/aging, as has been proposed for coronaviruses (
56). In this model, the RNA synthesizing activity of the RTC changes in time, possibly by proteolytic turnover of the replicase polyprotein. Moreover, were nsps generally contained within static networks, complementation between different (e.g., temperature sensitive) viruses carrying a mutation in one of these nsps would have to occur during the formation of these networks at the time of RTC/DMV assembly and would not be possible once these replicative structures had been assembled.
So far, live-cell imaging of viral infections has been limited mainly to the processes of entry and release of viral particles (
25,
42). Trafficking and the dynamics of viral replicative structures have received much less attention and for plus-strand RNA viruses have, so far, essentially been reported only for HCV (
74) and MHV (this study). Considering that these viruses belong to different virus families (the
Coronaviridae and the
Flaviviridae, respectively), the similarities observed between the two viruses are, at least in our opinion, quite remarkable. One feature is the occurrence of differently sized replicative structures, and we have now shown that the small but not the large ones traffic along microtubule tracks. Another, perhaps even more intriguing, feature is that in both cases structures appear to function as rigid entities. In view of the parallels observed between MHV and HCV, it is tempting to hypothesize that our findings reflect general features of the replication of plus-strand RNA viruses.