- freely available
Viruses 2012, 4(11), 3245-3269; doi:10.3390/v4113245
Abstract: Coronaviruses are positive-strand RNA viruses that are important infectious agents of both animals and humans. A common feature among positive-strand RNA viruses is their assembly of replication-transcription complexes in association with cytoplasmic membranes. Upon infection, coronaviruses extensively rearrange cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes to which the nonstructural proteins involved in RNA synthesis localize. Double-stranded RNA, presumably functioning as replicative intermediate during viral RNA synthesis, has been detected at the double-membrane vesicle interior. Recent studies have provided new insights into the assembly and functioning of the coronavirus replicative structures. This review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the replication-transcription complexes, and the location of viral RNA synthesis, with particular focus on the dynamics of the coronavirus replicative structures and individual replication-associated proteins.
Positive-strand RNA (+RNA) viruses are the most abundant viruses in nature. Many important pathogens belong to this category, including poliovirus (PV), hepatitis C virus (HCV) and the severe acute respiratory syndrome (SARS)-coronavirus (CoV). A distinctive common feature of +RNA viruses is the replication of their genomes in the cytoplasm of the host cell in association with rearranged cellular membranes that are remodeled into organelle-like membranous structures to which the viral replication-transcription complexes (RTCs) localize.
The various membrane rearrangements observed in +RNA virus-infected cells range in size from 40 to 400 nm, contain lipids that are derived from various cellular compartments and demonstrate an impressively diverse plethora of morphologies that include, among others, clusters of vesicles for the Picorna- and Togaviridae, spherule-like invaginations for the Bromoviridae and Nodaviridae, and vesicle packets and membranous webs for the Flaviviridae (reviewed in [1,2,3]). These membrane rearrangements seem to be beneficial for (i) sequestering and concentrating all viral and cellular components necessary for viral RNA synthesis and (ii) to provide a protective microenvironment against virus-elicited host defense mechanisms.
Also coronaviruses (CoVs), enveloped +RNA viruses that belong to the family Coronaviridae, extensively rearrange cellular membranes into organelle-like replicative structures during infection. These replicative structures consist of double membrane vesicles (DMVs) and convoluted membranes (CMs). The viral replicase proteins involved in RNA synthesis localize to both these structures, while double-stranded RNA (dsRNA), presumably functioning as replicative intermediate during viral RNA synthesis, has been detected at the DMV interior [4,5]. Recent studies have provided new insights into the assembly and functioning of the CoV replicative structures. This review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the RTCs, and the location of viral RNA synthesis, with particular focus on the dynamics of the coronavirus replicative structures and individual replication-associated proteins.
4. Membrane Rearrangements
4.1. Organelle-like Membranous Replicative Structures
Upon infection of host cells, CoVs induce a variety of membranous structures of which some have been associated with viral RNA synthesis. The first detectable membrane rearrangements in CoV-infected cells are 200 to 350 nm organelle-like structures that have been described for both MHV [47,62] and the SARS-CoV [5,63] and consist of spherical vesicles containing double lipid bilayers, termed DMVs (Figure 2). In between the clusters of DMVs, reticular CMs are characteristically present [4,62,64]. Later in infection large virion-containing vesicles (LVCVs) [4,5,62,65], highly organized cubic membrane structures [5,62] and condensed tubular bodies [62,64] are formed. The latter two structures are likely a result of the overexpression of CoV structural proteins during infection and do not seem to be involved in CoV replication .
Electron tomography studies demonstrated that in SARS-CoV infected cells the DMVs and CMs form an interconnected membranous network that is also continuous with the ER . This latter observation is in agreement with previous reports describing DMVs in close proximity to the ER or continuous with it [5,47,63]. Moreover, (partial) colocalization of replicase proteins together with the ER resident protein disulfide isomerase (PDI) has been reported , while also the translocon subunit Sec61α was found to be redistributed to the replicative structures upon SARS-CoV infection . The combined data indicate that the ER is the most likely membrane donor for the DMVs, despite the absence of most conventional ER markers on these structures [43,62,63,67].
The CoV replicative structures, i.e., DMVs and CMs (Figure 2), have been associated with viral RNA synthesis, as the MHV and SARS-CoV nsps have been shown to localize to these structures [4,5,19,43,59,62,63,68,69,70,71]. In addition, antibodies recognizing dsRNA, the presumed replicative intermediates, label the interior of the SARS-CoV-induced DMVs . Newly synthesized viral RNA, visualized by 5-bromouridine 5'-triphosphate (BrUTP) labeling, was observed in MHV-infected cells in close proximity to the replicative structures by immunoelectron microscopy [47,71].
4.2. Involvement of Cellular Pathways
Remodeling of eukaryotic cellular membranes into the replicative structures is likely dependent on the combined effects of both viral and cellular proteins and probably also on the specific lipid composition of the membranes themselves. Nevertheless, only few studies have been published addressing the involvement of cellular constituents in CoV replication and the generation of the replicative structures. It is conceivable that CoVs hijack cellular pathways to meet the conditions that are required for their replication, consequently adopting intrinsic properties of the utilized pathways themselves.
In agreement with the ER being the most likely membrane donor of the DMVs, an intimate association between the early secretory pathway and CoV replication has been demonstrated. RTC formation and replication in MHV-infected cells were inhibited when the secretory pathway was interfered with by blocking protein export at ER exit sites by treatment with the kinase inhibitor H89 or by expression of a dominant active Sar1 mutant . Also treatment with Brefeldin A (BFA), an inhibitor of ER-to-Golgi trafficking, or knockdown of its target GBF1, inhibited MHV replication while reducing the number of DMVs . Similar results were published for SARS-CoV infected cells treated with BFA and it was noticed that the inner and outer membranes of the DMVs were separated in BFA-treated cells , which may explain the observed inhibition of viral replication.
By their main ultrastructural characteristic, the double lipid bilayer, CoV DMVs very much resemble autophagosomes. This similarity prompted studies into the role of the autophagy machinery in CoV replication. Initial studies revealed a colocalization between the autophagosomal protein marker microtubule-associated protein light-chain 3 (LC3/Atg8) with the replicative structures ; moreover, viral replication was impaired and DMVs were not detected in the absence of the essential autophagy protein Atg5 . In other studies however, these colocalization data could not be reproduced , while the absence of Atg5 did not affect CoV replication [73,74]. Yet, LC3 was found in association with the replicative structures by Zhao and coworkers . Furthermore, others showed that, while the endogenous LC3 protein was recruited to the replicative structures, this was not the case for a GFP-tagged form of LC3 [69,75,76] that is often used as a marker for autophagosomes . In agreement herewith, CoV replication was shown to be unaffected in autophagy-deficient cells lacking Atg7, although depletion of LC3 severely affected CoV replication . Unlike autophagosomes, which may also be induced by expression of CoV nsp6 , CoV replicative structures were shown to be decorated with the non-lipidated form of LC3. Similar findings were also reported for EDEMosomes [78,79], ER-derived vesicles that transport ER chaperones to lysosomes. As the EDEMosome cargo proteins EDEM1 and OS-9 were also detected in association with the CoV replicative structures, it was proposed that CoVs hijack EDEMosomes for their replication [69,80]. In agreement herewith, the transmembrane SEL1L protein, which was recently shown to interact with LC3 and to have a critical function in EDEMosome biosynthesis, was also shown to colocalize with dsRNA foci in CoV-infected cells, while its depletion negatively affected CoV replication .
4.3. Role of Viral Proteins
The CoV nonstructural membrane proteins, nsp3, 4 and 6 probably play an essential role in the membrane rearrangements required for the induction of the replicative structures and in the anchoring of the RTCs to these structures. These proteins were shown to be engaged in homo- and heterotypic interactions . Interestingly, co-expression of nsp4 with the C-terminal one-third part of nsp3 (nsp3C) resulted in the relocalization of these proteins from the ER into discrete foci mostly localizing to the perinuclear region of the cell . Although nsp6 was not required for the observed relocalization, it was recruited to the perinuclear foci when co-expressed , in agreement with this protein interacting with nsp4 . Ultrastructural analysis of cells co-expressing nsp4 and nsp3C revealed that the membranes of the ER exhibited more curvature, although these membranes did not resemble the DMVs observed in CoV-infected cells , which may not be surprising as only the C-terminal part of nsp3 was used in these co-expressions with nsp4. Similar membrane rearrangements were not observed when the nsp3 from MHV was co-expressed with nsp4 from SARS-CoV (or vice versa), while (deletion) mutagenesis studies indicated essential roles for the large luminal loops of nsp3 and nsp4 in the relocalization of these proteins . While co-immunoprecipitation and immunofluorescence assays indicate that nsp3 and nsp4 of MHV interact, this interaction could not be confirmed using the Venus protein-fragment complementation assay . We hypothesize that the interactions between nsp3 and nsp4 mediate some kind of “zippering” of the lipid bilayers of the ER, which ultimately leads to the formation of the DMVs. In this model nsp3 and nsp4 interact via their luminal loops in such a way that their interaction prevents reconstitution of a functional Venus protein.
Several other studies also suggest an important role for nsp4 in the generation of the replicative structures. Disruption of the nsp4 glycosylation sites present in the loop between the first and second hydrophobic region, leads to the formation of aberrant DMVs in which the inner and outer membranes are detached, while the number of CMs is increased . In agreement herewith, although the fourth hydrophobic domain of nsp4 is dispensable, the other three transmembrane regions are required for CoV replication . Furthermore, co-expression of the counterparts of the CoV nsp3 and nsp4 of the distantly-related equine arteritis virus (EAV), resulted in the rearrangement of host cell membranes into DMVs, albeit with a morphology  differing from that observed in EAV-infected cells . Mutations of cysteine residues present in the luminal loop of EAV nsp3 (the EAV counterpart of CoV nsp4) resulted in altered morphologies of the DMVs, while the introduction of a N-glycosylation site in this loop also affected their morphology to some extent .
Like CoVs, other +RNA viruses also somehow induce membrane rearrangements that are required for their replication and transcription. For several of these viruses similar membrane rearrangements can be induced by the (co-)expression of nsps (for reviews see [1,2,3]). These nsps are either integral transmembrane proteins, examples being the NS4A proteins of Dengue virus (DENV)  and Kunjin virus  and the NS4B protein of hepatitis C virus (HCV) , or alternatively the proteins are only peripherally associated to the lipid bilayer, such as the 1a protein of brome mosaic virus (BMV)  and the 2C protein of poliovirus (PV) . Strikingly, however, also for the integral membrane proteins the occurrence of hydrophobic/amphipathic regions that do not span the lipid bilayer but are peripherally associated with membranes, which has also been demonstrated for CoV nsp3 and nsp6 [42,44,45], appears to be a common feature [88,93,94,95]. Another similarity among the nsps of different +RNA viruses appears to be their ability to assemble into larger protein complexes. Such interactions have been observed not only between nsp3, nsp4 and nsp6 of CoVs, but also between the nsps of other +RNA viruses, including the BMV 1a protein [96,97] and the flavivirus NS4A and NS4B proteins [98,99].
How are the nsps of CoVs able to induce the observed membrane rearrangements? We speculate that the similarities between the membrane-associated nsps of different +RNA viruses relate to their common ability to remodel membranes. The induction of membrane curvature in lipid bilayers is critical when remodeling host cellular membranes. The membrane-associated viral proteins may act as multimeric scaffolds able to impose such curvature, for instance by acting as wedges by inserting their amphipatic helices partially into one side of the bilayer (reviewed in [100,101,102]). The viral proteins may function similar to host cellular proteins known to induce membrane bending via a scaffold mechanism, as exemplified by the COPI and COPII complexes, and/or via the insertion of amphipathic domains into the lipid bilayer, as has been proposed for the small GTPase Sar1 (reviewed in [100,102]).
5. Dynamics of CoV Replicative Structures and Associated Proteins
The last few decades have provided virologists with exciting new information regarding the functions and structures of individual nsps and the characterization and formation of the membranous structures induced by +RNA viruses. These insights were obtained by classical biochemical, immunofluorescent and ultrastructural approaches. Unfortunately, little information regarding the dynamics of the (viral) proteins present at the membranous replicative structures in living cells is known. Such studies are important as classical approaches only provide static views of cellular processes and do not necessarily reflect the dynamics underlying virus replication in living cells. To date, only few studies on the dynamics of the replicative structures of plus-strand RNA viruses and their associated proteins have been published [103,104,105,106,107,108,109].
5.1. Dynamics of the CoV Replicative Structures
By using a GFP-tagged version of nsp2, shown by immuno-electron microscopy (IEM) to be recruited to the DMVs and CMs, as a marker the dynamics of the CoV replicative structures were studied using live cell imaging . These studies showed for the first time that the CoV replicative structures are moving through the cell. However, it appeared that the CoV replicative structures consist of two classes that demonstrate different mobilities: large structures lacking any displacement and smaller structures with relatively high saltatory mobility. The smaller replicative structures were hypothesized to correspond to individual DMVs, while the larger ones supposedly represent the DMV/CM assemblies that have been observed in ultrastructural studies of CoV-infected cells [4,62]. In SARS-CoV-infected cells the DMVs are confined to a reticulovesicular network . We therefore speculate that the small structures have not yet been ‘captured’ into this network. However, correlative light-electron microscopy studies will be required to solve this issue.
Live cell imaging studies of HCV- and Semliki Forest virus (SFV)-infected cells also reveal the presence of replicative structures that could be discriminated on the basis of their size and mobility. HCV induces the formation of a so-called membranous web, in which DMVs can be observed . Large HCV structures, probably representing membranous webs, exhibited limited movement, whereas smaller ones were mobile and could travel long distances throughout the cytoplasm . SFV-induced vacuoles are assembled at the plasma membrane after which they are transported to modified lysosomes [106,107]. SFV-infected cells also harbor large acidic immobile perinuclear vesicles and smaller acidic cytoplasmic vesicles that showed saltatory movements. In addition to these acidic vesicles, SFV-infected cells contain a class of non-acidic highly mobile vesicles that displayed multidirectional short-distance movements. Moreover, fusion of the neutral mobile structures with the acidic mobile structures resulted in the formation of the large acidic structures . Such events of fusion of smaller replicative structures into larger ones have not been observed (yet) for the CoV and HCV replicative structures [108,110].
The calculated velocities of the saltatory movements of the smaller nsp2-positive structures in CoV-infected cells  correspond to those measured for microtubule-mediated transport . The observed association of these structures with microtubules and the inhibition of trafficking in the presence of a microtubule network-disturbing drug confirmed the transport of the smaller structures on microtubular tracks . A role of the cytoskeleton in the movement of replicative structures has also been described for HCV, SFV, and PV [105,107,108,109]. Disruption of a functional microtubular network inhibited the movement of the small HCV and nascent PV replicative structures [108,109] and the trafficking of SFV neutral vesicles to acidic organelles , concomitant with dispersal of these structures throughout the cytoplasm. Strikingly, inhibition of microtubule-dependent trafficking did not or only modestly affect the replication of these viruses [109,110,112]. Disruption of the actin network also did not affect CoV replication much, although the replicative structures again failed to accumulate in the perinuclear area . Collectively, these results show that the cytoskeleton is required for the perinuclear accumulation of the replicative structure rather than for replication per se. Additional studies are required to clarify the role of the perinuclear targeting of the CoV replicative structures during infection.
5.2. Mobility of Replicative Structure-Associated Proteins
Up till now, only few studies have addressed the dynamics of individual viral proteins when present at the replicative structures. Recently, we analyzed the dynamic properties of three replication-associated proteins, i.e., the soluble nsp2 and N proteins and the integral membrane protein nsp4, and demonstrated that these proteins display different diffusional mobilities when present at the replicative structures.
Nsp2, when expressed in trans is recruited to the replicative structures . After recruitment, nsp2 was shown to be immobilized by using fluorescent recovery after photobleaching (FRAP) methodology. In other words, nsp2 already associated with the replicative structures was not exchanged by nsp2 present at other locations in the cell. Similar results have been demonstrated for other +RNA virus replication-associated proteins, although the published literature on this subject is limited. Also in HCV-infected cells, NS5A-positive structures showed a static internal architecture when (part of) the NS5A fluorescent protein pool was bleached . When expressed individually, NS5A is highly mobile , similar to nsp2. Apparently, in the context of a viral infection, when other viral proteins are present, MHV nsp2 and HCV NS5A are immobilized at the replicative structures presumably due to protein-RNA or protein-protein interactions. In agreement herewith, large-scale protein-protein interaction studies demonstrated that nsp2 of MHV and SARS-CoV is engaged in a multitude of interactions with itself, nsp3, nsp4, nsp6, nsp7, nsp8, nsp11, nsp15 and nsp16 [82,114,115,116]. These observations are remarkable in view of the dispensability of nsp2 during CoV infection in vitro . Yet, nsp2 has to be somehow important in vivo as its coding sequence is maintained during evolution.
In addition to its structural role in the coronavirion, i.e., in packaging of the genomic RNA into the RNP complex, the structural N protein is also important in coronavirus replication and has been detected in the perinuclear region of infected cells colocalizing with markers for the replicative structures [62,67,117,118]. The interaction of the N protein with nsp3 is presumably important for the initial recruitment of N to the replicative structures , while N-N protein interactions were sufficient for recruitment of (mutant) N proteins in the presence of wild type N proteins . In contrast to nsp2, the N protein is not immobilized at the CoV replicative structures but is associated with it rather dynamically . This may not be surprising, as the multifunctional N protein is both involved in viral replication [121,122,123] and virion assembly . As the CoV replicative structures and virion assembly sites appear to be spatially separated, the newly synthesized genomic viral RNA needs to be transported from the replication sites to the assembly sites. The N protein presumably facilitates this transport, consistent with its dynamic behavior.
When expressed in trans in infected cells, the nsp4-GFP fusion protein was detected at the ER and at the replicative structures [43,81]. By performing fluorescence loss in photobleaching (FLIP) experiments continuity was demonstrated between the membranes of the ER and the replicative structures that harbor nsp4 , in agreement with the model that the DMVs and CMs form an interconnected network that is continuous with the ER . However, nsp4 displayed different diffusional mobilities at different subcellular locations . It was more mobile in the ER than at the replicative structures. This reduced mobility may be (partly) due to its engagement in interactions with the other transmembrane-containing nsps as well as with itself . Also the mobility of the HCV NS4B protein, which is an integral transmembrane protein as well, depends on its intracellular location. When expressed in the absence of other viral proteins, this protein is present at the ER and at so-called membrane-associated foci (MAFs) that are induced upon expression of this protein . FRAP analysis showed that NS4B present at the MAFs had a reduced mobility compared to NS4B at the ER, which was suggested to result from NS4B being engaged in different interactions when present on MAFs or the ER .
6. CoV Replicative Structures and RNA Synthesis
Currently, one of the most enigmatic issues regarding CoV replication is the precise localization of the sites of active viral RNA synthesis. Although CoV RNA synthesis appears to be protected by membranes  it is still unclear whether synthesis of nascent viral RNA occurs at sites of dsRNA accumulation, as pores connecting the interior of the coronavirus DMVs with the cytoplasm have not been detected , while the nsps localize to both DMVs and CMs [4,5,19,43,59,62,63,68,69,70,71]. To identify sites of nascent viral RNA synthesis, one has to define what actually constitutes the active viral replication complexes. Although dsRNA molecules function as intermediates of replication and transcription, their presence at certain sites per se does not imply (all) these structures to be actively involved in RNA synthesis. Likewise, the location of viral enzymes that are required for RNA synthesis does not need to correlate with active RTCs. Moreover, newly synthesized RNAs are not necessarily located at their site of synthesis as they may diffuse or be transported away to other subcellular locations. In view of these considerations, sites active in RNA synthesis are expected to contain at least three components: the RdRp, dsRNA intermediates active in replication/transcription and nascent viral RNA.
Newly synthesized viral RNAs, visualized by 5-bromouridine 5’-triphosphate (BrUTP) labeling, were shown to colocalize with antibodies recognizing either nsp5 or the C-terminal part of pp1a , and were observed in close proximity to the DMVs by immunoelectron microscopy in MHV-infected cells [47,71]. Recently, a new method was used to detect and visualize newly synthesized coronaviral RNA by incorporation of an alkyne-modified uridine analog, 5-ethynyl uridine (EU), onto which an azide-derivatized fluophore was coupled via a copper (I)-catalyzed cycloaddition reaction (click chemistry). With this method, it was shown that throughout MHV infection foci of nascent RNAs could be detected, which colocalize with the RdRp-containing nsp12, indicating that they correspond with sites of active coronaviral RNA synthesis. The relationship between nascent RNA and dsRNA is, however, less clear. While early in infection nascent RNAs colocalize at or adjacent to patches of dsRNA dots, presumably corresponding to DMVs, this correlation is much less apparent at later times when the dsRNA dots are spread throughout the cell. Many dsRNA dots are apparently not transcriptionally active as no EU labeling was associated with them, while many foci of EU labeling did not appear to colocalize with the dsRNA dots .
Different models can be put forward to explain these observations. In one model, DMVs function as the sites of active RNA synthesis. At the later times in infection, many DMVs are no longer active, while the ones that are active may contain only little dsRNA, resulting in less apparent colocalization of dsRNA and EU labeling. In another model, DMVs are non-functional end-stage products. They are not actively involved in RNA synthesis, but rather harbor dsRNAs that are not (longer) functioning as intermediates in RNA synthesis. In this model, which is in agreement with the presumed absence of pores in these structures , the CMs would be the only plausible alternative for the sites of active RNA synthesis. In yet another model, DMVs may be the initial sites of active RNA synthesis, particularly early in infection, while at later times the membranes become sealed, connections are lost and RNA synthesis shifts to the CM assemblies. Clearly, ultrastructural studies will be required, ideally (co)localizing nascent RNAs as well as the RdRp, to definitely determine the precise localization of CoV RNA synthesis.
For most other +RNA viruses the identification of the sites of active RNA synthesis appears less complicated. Nascent RNAs, as well as nsps, have been shown to label the spherules that are observed in BMV- , SFV-  or flock house virus (FHV)-  infected cells at the modified ER, lysosomes and mitochondria, respectively, indicating that these structures correspond with the sites of active RNA synthesis. Nascent RNAs were previously also shown to colocalize with dsRNA in Kunjin flavivirus-infected cells . Electron tomography of the membrane rearrangements observed in flavirus-infected cells revealed that the inner content of the DMVs, which contains nsps and dsRNA, is connected to the cytoplasm via a pore [129,130], indicating that these DMVs are actually spherule-like invaginations (once) active in RNA synthesis.
7. Future Directions
CoV replicative structures/RTCs are macromolecular assemblies, the components of which are engaged in a plethora of protein-protein, protein-RNA, and protein-lipid interactions [114,115,116,131]. Currently, the exact composition of the replicative structures/RTCs is not known, let alone the full arsenal of interactions occurring within these structures. Moreover, the replicative structures are likely to be subject to some form of maturation [14,19,20,113], as their composition appears to change during the course of infection as determined by biochemical and immunofluorescence analyses. Thus, it will be of interest to confirm, extend and refine the previously published protein-protein interactome studies that have been published for SARS-CoV [114,115,116], for example by investigating protein-protein interactions of other CoVs using novel (large-scale) screening approaches. Likewise, it will be of interest to get more insight into the involvement of host proteins in the formation of these structures, for example by screening for host proteins that interact with the CoV nsps or by elucidating the protein content of purified replicative structures -preferably in time- using mass spectrometry.
One of the difficulties associated with these studies is the complexity of discriminating whether host proteins are directly involved in the formation of the replicative structures themselves or in RNA synthesis per se, as inhibition of either process will result in reduced RNA synthesis, protein expression and DMV formation. Therefore, assays are needed in which the formation of replicative structures can be studied independent of viral replication. Such assays may be provided by co-expression of viral proteins [81,85] that induce the rearrangement of cellular membranes. Recently, by using such a replication-independent assay, reticulons, which form a family of ER membrane-shaping proteins, were implicated in the formation of the BMV-induced, ER-derived spherules that are associated with viral RNA synthesis . Reticulons are involved in the induction of ER membrane curvature [133,134,135] and seem to be required by BMV for determining the size of the spherules and for stabilizing the spherule necks . It will be of interest to study the putative role of these proteins in CoV replication and in reshaping of the ER membranes by CoV nsps. Other proteins playing a role in shaping and remodeling of the ER are the atlastins and CLIMP-63 [133,136]. Also these proteins are putative candidates involved in CoV-induced membrane remodeling and would deserve to be investigated.
In addition to the role of virus and host proteins in formation of the replicative structures, lipids may also be important in this process. Dynamic alteration of the lipid composition can induce curvature of membranes to some extent [100,102,137,138], which is, however, unlikely to be sufficient to generate the organelle-like structures observed in +RNA virus-infected cells. It is more likely that lipids, together with lipid-modifying enzymes, contribute to the formation of the replicative structures by providing a suitable microenvironment to which the viral (and cellular) membrane shaping proteins are recruited. Even more, +RNA viruses may specifically hijack lipid-modifying enzymes for their own advantage [139,140]. This is underscored by several studies, which show that inhibition of lipid synthesis by using either drugs or small interfering RNAs dramatically affects the replication of +RNA viruses [139,141,142,143,144]. It will be of interest to study how and to what extent CoV infection affects the lipid homeostasis in infected cells, for example by analyzing the lipid repertoire of infected cells by lipidomics techniques.
Finally, as all processes occurring in living cells are inherently dynamic in nature, it is also desirable to get more insight into the biogenesis and functioning of the replicative structures using various live cell imaging approaches. For example, photoactivatable fluorescent proteins can be used to investigate the formation of the replicative structures in real-time by ‘optical pulse-labeling’ in living cells, while the behavior of individual nsps associated with the RTCs can be studied by selectively ‘switching-on’ (sub)populations of proteins [145,146]. Furthermore, tagging of viral RNA by genetic incorporation of specific RNA sequences that bind fluorescently-tagged RNA-binding proteins [147,148], hybridization of fluorescent ‘molecular beacons’ to the viral RNAs , or combining metabolic labeling of viral RNAs with Cu-independent click chemistry , will allow visualization and tracking of these ribonucleic acid species in living cells. Concomitant live cell imaging of viral RNA and the N proteins may be used to investigate the transport of nascent RNA from the replication to the assembly sites. In addition, super resolution microscopy techniques, like photoactivated localization microscopy (PALM), stimulated emission depletion microscsopy (STED) and stochastic optical reconstruction microscopy (STORM) [151,152,153] can all be applied to investigate the CoV RTCs and the membranous replicative structures at the ultrastructural level using fluorescently-tagged proteins, while EU-labeling of viral RNA in combination with correlative light-electron microscopy may provide the resolution to indisputably pinpoint the exact location of viral RNA synthesis .
We would like to thank Oliver Wicht and Christine Burkhard from the Virology Division, Faculty of Veterinary Medicine, Utrecht University for stimulating discussion and Fulvio Reggiori and Mustafa Ulasli from the Department of Cell Biology and Cell Microscopy Center, University Medical Centre Utrecht for providing the EM pictures of the CoV replicative structures. C.A.M.D.H. is supported by grants from the Netherlands Organization for Scientific Research (NWO-ALW) and Utrecht University (High Potential).
Conflict of Interest
The authors declare no conflict of interest.
References and Notes
- den Boon, J.A.; Ahlquist, P. Organelle-Like Membrane Compartmentalization of Positive-Strand RNA Virus Replication Factories. Annu. Rev. Microbiol. 2010, 64, 241–256. [Google Scholar] [CrossRef]
- den Boon, J.A.; Diaz, A.; Ahlquist, P. Cytoplasmic Viral Replication Complexes. Cell Host Microbe 2010, 8, 77–85. [Google Scholar] [CrossRef]
- Miller, S.; Krijnse-Locker, J. Modification of Intracellular Membrane Structures for Virus Replication. Nat. Rev. Microbiol. 2008, 6, 363–374. [Google Scholar] [CrossRef]
- Knoops, K.; Kikkert, M.; van den Worm, S.H.E.; Zevenhoven-Dobbe, J.C.; van der Meer, Y.; Koster, A.J.; Mommaas, A.M.; Snijder, E.J. SARS-Coronavirus Replication is Supported by a Reticulovesicular Network of Modified Endoplasmic Reticulum. PLoS Biol. 2008, 6, 1957–1974. [Google Scholar]
- Goldsmith, C.S.; Tatti, K.M.; Ksiazek, T.G.; Rollin, P.E.; Comer, J.A.; Lee, W.W.; Rota, P.A.; Bankamp, B.; Bellini, W.J.; Zaki, S.R. Ultrastructural Characterization of SARS Coronavirus. Emerg. Infect. Dis. 2004, 10, 320–326. [Google Scholar]
- Drosten, C.; Günther, S.; Preiser, W.; van der Werf, S.; Brodt, H.R.; Becker, S.; Rabenau, H.; Panning, M.; Kolesnikova, L.; Fouchier, R.A.M.; et al. Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome. N. Engl. J. Med. 2003, 348, 1967–1976. [Google Scholar] [CrossRef]
- Ksiazek, T.G.; Erdman, D.; Goldsmith, C.S.; Zaki, S.R.; Peret, T.; Emery, S.; Tong, S.; Urbani, C.; Comer, J.A.; Lim, W.; et al. A Novel Coronavirus Associated with Severe Acute Respiratory Syndrome. N. Engl. J. Med. 2003, 348, 1953–1966. [Google Scholar] [CrossRef]
- Guan, Y.; Zheng, B.J.; He, Y.Q.; Liu, X.L.; Zhuang, Z.X.; Cheung, C.L.; Luo, S.W.; Li, P.H.; Zhang, L.J.; Guan, Y.J.; et al. Isolation and Characterization of Viruses Related to the SARS Coronavirus from Animals in Southern China. Science 2003, 302, 276–278. [Google Scholar] [CrossRef]
- Li, W.; Shi, Z.; Yu, M.; Ren, W.; Smith, C.; Epstein, J.H.; Wang, H.; Crameri, G.; Hu, Z.; Zhang, H.; et al. Bats are Natural Reservoirs of SARS-Like Coronaviruses. Science 2005, 310, 676–679. [Google Scholar]
- Gorbalenya, A.E.; Enjuanes, L.; Ziebuhr, J.; Snijder, E.J. Nidovirales: Evolving the Largest RNA Virus Genome. Virus Res. 2006, 117, 17–37. [Google Scholar] [CrossRef]
- Sawicki, S.G.; Sawicki, D.L.; Siddell, S.G. A Contemporary View of Coronavirus Transcription. J. Virol. 2007, 81, 20–29. [Google Scholar] [CrossRef]
- Sawicki, S.G.; Sawicki, D.L. Coronaviruses use Discontinuous Extension for Synthesis of Subgenome-Length Negative Strands. Adv. Exp. Med. Biol. 1995, 380, 499–506. [Google Scholar] [CrossRef]
- Zuniga, S.; Sola, I.; Alonso, S.; Enjuanes, L. Sequence Motifs Involved in the Regulation of Discontinuous Coronavirus Subgenomic RNA synthesis. J. Virol. 2004, 78, 980–994. [Google Scholar] [CrossRef]
- Sawicki, S.G.; Sawicki, D.L. Coronavirus Minus-Strand RNA Synthesis and Effect of Cycloheximide on Coronavirus RNA Synthesis. J. Virol. 1986, 57, 328–334. [Google Scholar]
- Sawicki, D.; Wang, T.; Sawicki, S. The RNA Structures Engaged in Replication and Transcription of the A59 Strain of Mouse Hepatitis Virus. J. Gen. Virol. 2001, 82, 385–396. [Google Scholar]
- Bredenbeek, P.J.; Pachuk, C.J.; Noten, A.F.; Charite, J.; Luytjes, W.; Weiss, S.R.; Spaan, W.J. The Primary Structure and Expression of the Second Open Reading Frame of the Polymerase Gene of the Coronavirus MHV-A59; a Highly Conserved Polymerase is Expressed by an Efficient Ribosomal Frameshifting Mechanism. Nucleic Acids Res. 1990, 18, 1825–1832. [Google Scholar]
- Brierley, I.; Digard, P.; Inglis, S.C. Characterization of an Efficient Coronavirus Ribosomal Frameshifting Signal: Requirement for an RNA Pseudoknot. Cell 1989, 57, 537–547. [Google Scholar] [CrossRef]
- Ziebuhr, J.; Snijder, E.J.; Gorbalenya, A.E. Virus-Encoded Proteinases and Proteolytic Processing in the Nidovirales. J. Gen. Virol. 2000, 81, 853–879. [Google Scholar]
- Deming, D.J.; Graham, R.L.; Denison, M.R.; Baric, R.S. Processing of Open Reading Frame 1a Replicase Proteins nsp7 to nsp10 in Murine Hepatitis Virus Strain A59 Replication. J. Virol. 2007, 81, 10280–10291. [Google Scholar]
- Sawicki, S.G.; Sawicki, D.L.; Younker, D.; Meyer, Y.; Thiel, V.; Stokes, H.; Siddell, S.G. Functional and Genetic Analysis of Coronavirus Replicase-Transcriptase Proteins. PLoS Pathog. 2005, 1, e39. [Google Scholar] [CrossRef]
- te Velthuis, A.J.; Arnold, J.J.; Cameron, C.E.; van den Worm, S.H.; Snijder, E.J. The RNA Polymerase Activity of SARS-Coronavirus nsp12 is Primer Dependent. Nucleic Acids Res. 2010, 38, 203–214. [Google Scholar]
- Imbert, I.; Guillemot, J.C.; Bourhis, J.M.; Bussetta, C.; Coutard, B.; Egloff, M.P.; Ferron, F.; Gorbalenya, A.E.; Canard, B. A Second, Non-Canonical RNA-Dependent RNA Polymerase in SARS Coronavirus. EMBO J. 2006, 25, 4933–4942. [Google Scholar]
- Zhai, Y.; Sun, F.; Li, X.; Pang, H.; Xu, X.; Bartlam, M.; Rao, Z. Insights into SARS-CoV Transcription and Replication from the Structure of the nsp7-nsp8 Hexadecamer. Nat. Struct. Mol. Biol. 2005, 12, 980–986. [Google Scholar]
- Xiao, Y.; Ma, Q.; Restle, T.; Shang, W.; Svergun, D.I.; Ponnusamy, R.; Sczakiel, G.; Hilgenfeld, R. Nonstructural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer that Exhibits Primer-Independent RNA Polymerase Activity. J. Virol. 2012, 86, 4444–4454. [Google Scholar] [CrossRef]
- te Velthuis, A.J.; van den Worm, S.H.; Snijder, E.J. The SARS-Coronavirus nsp7+nsp8 Complex is a Unique Multimeric RNA Polymerase Capable of both De Novo Initiation and Primer Extension. Nucleic Acids Res. 2012, 40, 1737–1747. [Google Scholar] [CrossRef]
- Seybert, A.; Posthuma, C.C.; van Dinten, L.C.; Snijder, E.J.; Gorbalenya, A.E.; Ziebuhr, J. A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases. J. Virol. 2005, 79, 696–704. [Google Scholar] [CrossRef]
- Seybert, A.; Hegyi, A.; Siddell, S.G.; Ziebuhr, J. The Human Coronavirus 229E Superfamily 1 Helicase has RNA and DNA Duplex-Unwinding Activities with 5'-to-3' Polarity. RNA 2000, 6, 1056–1068. [Google Scholar] [CrossRef]
- Tanner, J.A.; Watt, R.M.; Chai, Y.B.; Lu, L.Y.; Lin, M.C.; Peiris, J.S.; Poon, L.L.; Kung, H.F.; Huang, J.D. The Severe Acute Respiratory Syndrome (SARS) Coronavirus NTPase/helicase Belongs to a Distinct Class of 5' to 3' Viral Helicases. J. Biol. Chem. 2003, 278, 39578–39582. [Google Scholar]
- Ivanov, K.A.; Thiel, V.; Dobbe, J.C.; van der Meer, Y.; Snijder, E.J.; Ziebuhr, J. Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase. J. Virol. 2004, 78, 5619–5632. [Google Scholar] [CrossRef]
- Ivanov, K.A.; Ziebuhr, J. Human Coronavirus 229E Nonstructural Protein 13: Characterization of Duplex-Unwinding, Nucleoside Triphosphatase, and RNA 5'-Triphosphatase Activities. J. Virol. 2004, 78, 7833–7838. [Google Scholar] [CrossRef]
- Chen, Y.; Cai, H.; Pan, J.; Xiang, N.; Tien, P.; Ahola, T.; Guo, D. Functional Screen Reveals SARS Coronavirus Nonstructural Protein nsp14 as a Novel Cap N7 Methyltransferase. Proc. Natl. Acad. Sci. USA 2009, 106, 3484–3489. [Google Scholar]
- Decroly, E.; Imbert, I.; Coutard, B.; Bouvet, M.; Selisko, B.; Alvarez, K.; Gorbalenya, A.E.; Snijder, E.J.; Canard, B. Coronavirus Nonstructural Protein 16 is a Cap-0 Binding Enzyme Possessing (Nucleoside-2'O)-Methyltransferase Activity. J. Virol. 2008, 82, 8071–8084. [Google Scholar] [CrossRef]
- Bouvet, M.; Debarnot, C.; Imbert, I.; Selisko, B.; Snijder, E.J.; Canard, B.; Decroly, E. In Vitro Reconstitution of SARS-Coronavirus mRNA Cap Methylation. PLoS Pathog. 2010, 6, e1000863. [Google Scholar]
- Lugari, A.; Betzi, S.; Decroly, E.; Bonnaud, E.; Hermant, A.; Guillemot, J.C.; Debarnot, C.; Borg, J.P.; Bouvet, M.; Canard, B. et al. Molecular Mapping of the RNA Cap 2'-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16. J. Biol. Chem. 2010, 285, 33230–33241. [Google Scholar]
- Minskaia, E.; Hertzig, T.; Gorbalenya, A.E.; Campanacci, V.; Cambillau, C.; Canard, B.; Ziebuhr, J. Discovery of an RNA Virus 3'→5' Exoribonuclease that is Critically Involved in Coronavirus RNA Synthesis. Proc. Natl. Acad. Sci. USA 2006, 103, 5108–5113. [Google Scholar]
- Ivanov, K.A.; Hertzig, T.; Rozanov, M.; Bayer, S.; Thiel, V.; Gorbalenya, A.E.; Ziebuhr, J. Major Genetic Marker of Nidoviruses Encodes a Replicative Endoribonuclease. Proc. Natl. Acad. Sci. USA 2004, 101, 12694–12699. [Google Scholar]
- Bhardwaj, K.; Sun, J.; Holzenburg, A.; Guarino, L.A.; Kao, C.C. RNA Recognition and Cleavage by the SARS Coronavirus Endoribonuclease. J. Mol. Biol. 2006, 361, 243–256. [Google Scholar]
- Eckerle, L.D.; Becker, M.M.; Halpin, R.A.; Li, K.; Venter, E.; Lu, X.; Scherbakova, S.; Graham, R.L.; Baric, R.S.; Stockwell, T.B.; et al. Infidelity of SARS-CoV Nsp14-Exonuclease Mutant Virus Replication is Revealed by Complete Genome Sequencing. PLoS Pathog. 2010, 6, e1000896. [Google Scholar] [CrossRef]
- Denison, M.R.; Graham, R.L.; Donaldson, E.F.; Eckerle, L.D.; Baric, R.S. Coronaviruses: An RNA Proofreading Machine Regulates Replication Fidelity and Diversity. RNA Biol. 2011, 8, 270–279. [Google Scholar] [CrossRef]
- Bouvet, M.; Imbert, I.; Subissi, L.; Gluais, L.; Canard, B.; Decroly, E. RNA 3'-End Mismatch Excision by the Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein nsp10/nsp14 Exoribonuclease Complex. Proc. Natl. Acad. Sci. USA 2012, 109, 9372–9377. [Google Scholar]
- Harcourt, B.H.; Jukneliene, D.; Kanjanahaluethai, A.; Bechill, J.; Severson, K.M.; Smith, C.M.; Rota, P.A.; Baker, S.C. Identification of Severe Acute Respiratory Syndrome Coronavirus Replicase Products and Characterization of Papain-Like Protease Activity. J. Virol. 2004, 78, 13600–13612. [Google Scholar] [CrossRef]
- Oostra, M.; Hagemeijer, M.C.; van Gent, M.; Bekker, C.P.; te Lintelo, E.G.; Rottier, P.J.; de Haan, C.A. Topology and Membrane Anchoring of the Coronavirus Replication Complex: Not all Hydrophobic Domains of nsp3 and nsp6 are Membrane Spanning. J. Virol. 2008, 82, 12392–12405. [Google Scholar] [CrossRef]
- Oostra, M.; te Lintelo, E.G.; Deijs, M.; Verheije, M.H.; Rottier, P.J.; de Haan, C.A. Localization and Membrane Topology of Coronavirus Nonstructural Protein 4: Involvement of the Early Secretory Pathway in Replication. J. Virol. 2007, 81, 12323–12336. [Google Scholar] [CrossRef]
- Kanjanahaluethai, A.; Chen, Z.; Jukneliene, D.; Baker, S.C. Membrane Topology of Murine Coronavirus Replicase Nonstructural Protein 3. Virology 2007, 361, 391–401. [Google Scholar] [CrossRef]
- Baliji, S.; Cammer, S.A.; Sobral, B.; Baker, S.C. Detection of Nonstructural Protein 6 in Murine Coronavirus-Infected Cells and Analysis of the Transmembrane Topology by using Bioinformatics and Molecular Approaches. J. Virol. 2009, 83, 6957–6962. [Google Scholar] [CrossRef]
- Kanjanahaluethai, A.; Baker, S.C. Identification of Mouse Hepatitis Virus Papain-like Proteinase 2 Activity. J. Virol. 2000, 74, 7911–7921. [Google Scholar]
- Gosert, R.; Kanjanahaluethai, A.; Egger, D.; Bienz, K.; Baker, S.C. RNA Replication of Mouse Hepatitis Virus Takes Place at Double-Membrane Vesicles. J. Virol. 2002, 76, 3697–3708. [Google Scholar]
- Sutton, G.; Fry, E.; Carter, L.; Sainsbury, S.; Walter, T.; Nettleship, J.; Berrow, N.; Owens, R.; Gilbert, R.; Davidson, A.; et al. The nsp9 Replicase Protein of SARS-Coronavirus, Structure and Functional Insights. Structure 2004, 12, 341–353. [Google Scholar]
- Su, D.; Lou, Z.; Sun, F.; Zhai, Y.; Yang, H.; Zhang, R.; Joachimiak, A.; Zhang, X.C.; Bartlam, M.; Rao, Z. Dodecamer Structure of Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein nsp10. J. Virol. 2006, 80, 7902–7908. [Google Scholar] [CrossRef]
- Zust, R.; Cervantes-Barragan, L.; Kuri, T.; Blakqori, G.; Weber, F.; Ludewig, B.; Thiel, V. Coronavirus Non-Structural Protein 1 is a Major Pathogenicity Factor: Implications for the Rational Design of Coronavirus Vaccines. PLoS Pathog. 2007, 3, e109. [Google Scholar]
- Wang, G.; Chen, G.; Zheng, D.; Cheng, G.; Tang, H. PLP2 of Mouse Hepatitis Virus A59 (MHV-A59) Targets TBK1 to Negatively Regulate Cellular Type I Interferon Signaling Pathway. PLoS One 2011, 6, e17192. [Google Scholar]
- Wathelet, M.G.; Orr, M.; Frieman, M.B.; Baric, R.S. Severe Acute Respiratory Syndrome Coronavirus Evades Antiviral Signaling: Role of nsp1 and Rational Design of an Attenuated Strain. J. Virol. 2007, 81, 11620–11633. [Google Scholar] [CrossRef]
- Narayanan, K.; Huang, C.; Lokugamage, K.; Kamitani, W.; Ikegami, T.; Tseng, C.T.; Makino, S. Severe Acute Respiratory Syndrome Coronavirus nsp1 Suppresses Host Gene Expression, Including that of Type I Interferon, in Infected Cells. J. Virol. 2008, 82, 4471–4479. [Google Scholar]
- Kuri, T.; Eriksson, K.K.; Putics, A.; Zust, R.; Snijder, E.J.; Davidson, A.D.; Siddell, S.G.; Thiel, V.; Ziebuhr, J.; Weber, F. The ADP-Ribose-1"-Monophosphatase Domains of SARS-Coronavirus and Human Coronavirus 229E Mediate Resistance to Antiviral Interferon Responses. J. Gen. Virol. 2011, 92, 1899–1905. [Google Scholar]
- Kamitani, W.; Narayanan, K.; Huang, C.; Lokugamage, K.; Ikegami, T.; Ito, N.; Kubo, H.; Makino, S. Severe Acute Respiratory Syndrome Coronavirus nsp1 Protein Suppresses Host Gene Expression by Promoting Host mRNA Degradation. Proc. Natl. Acad. Sci. USA 2006, 103, 12885–12890. [Google Scholar]
- Kamitani, W.; Huang, C.; Narayanan, K.; Lokugamage, K.G.; Makino, S. A Two-Pronged Strategy to Suppress Host Protein Synthesis by SARS Coronavirus Nsp1 Protein. Nat. Struct. Mol. Biol. 2009, 16, 1134–1140. [Google Scholar]
- Chen, C.J.; Sugiyama, K.; Kubo, H.; Huang, C.; Makino, S. Murine Coronavirus Nonstructural Protein p28 Arrests Cell Cycle in G0/G1 Phase. J. Virol. 2004, 78, 10410–10419. [Google Scholar]
- Gadlage, M.J.; Graham, R.L.; Denison, M.R. Murine Coronaviruses Encoding nsp2 at Different Genomic Loci have Altered Replication, Protein Expression, and Localization. J. Virol. 2008, 82, 11964–11969. [Google Scholar] [CrossRef]
- Graham, R.L.; Sims, A.C.; Brockway, S.M.; Baric, R.S.; Denison, M.R. The nsp2 Replicase Proteins of Murine Hepatitis Virus and Severe Acute Respiratory Syndrome Coronavirus are Dispensable for Viral Replication. J. Virol. 2005, 79, 13399–13411. [Google Scholar]
- Perlman, S.; Netland, J. Coronaviruses Post-SARS: Update on Replication and Pathogenesis. Nat. Rev. Microbiol. 2009, 7, 439–450. [Google Scholar] [CrossRef]
- Ziebuhr, J. The Coronavirus Replicase: Insights into a Sophisticated Enzyme Machinery. Adv. Exp. Med. Biol. 2006, 581, 3–11. [Google Scholar] [CrossRef]
- Ulasli, M.; Verheije, M.H.; de Haan, C.A.; Reggiori, F. Qualitative and Quantitative Ultrastructural Analysis of the Membrane Rearrangements Induced by Coronavirus. Cell. Microbiol. 2010, 12, 844–861. [Google Scholar] [CrossRef]
- Snijder, E.J.; van der Meer, Y.; Zevenhoven-Dobbe, J.; Onderwater, J.J.; van der Meulen, J.; Koerten, H.K.; Mommaas, A.M. Ultrastructure and Origin of Membrane Vesicles Associated with the Severe Acute Respiratory Syndrome Coronavirus Replication Complex. J. Virol. 2006, 80, 5927–5940. [Google Scholar] [CrossRef]
- David-Ferreira, J.F.; Manaker, R.A. An Electron Microscope Study of the Development of a Mouse Hepatitis Virus in Tissue Culture Cells. J. Cell. Biol. 1965, 24, 57–78. [Google Scholar] [CrossRef]
- Ng, M.L.; Tan, S.H.; See, E.E.; Ooi, E.E.; Ling, A.E. Proliferative Growth of SARS Coronavirus in Vero E6 Cells. J. Gen. Virol. 2003, 84, 3291–3303. [Google Scholar]
- Knoops, K.; Swett-Tapia, C.; van den Worm, S.H.E.; te Velthuis, A.J.W.; Koster, A.J.; Mommaas, A.M.; Snijder, E.J.; Kikkert, M. Integrity of the Early Secretory Pathway Promotes, but is Not Required for, Severe Acute Respiratory Syndrome Coronavirus RNA Synthesis and Virus-Induced Remodeling of Endoplasmic Reticulum Membranes. J. Virol. 2010, 84, 833–846. [Google Scholar]
- Verheije, M.H.; Raaben, M.; Mari, M.; Te Lintelo, E.G.; Reggiori, F.; van Kuppeveld, F.J.; Rottier, P.J.; de Haan, C.A. Mouse Hepatitis Coronavirus RNA Replication Depends on GBF1-Mediated ARF1 Activation. PLoS Pathog. 2008, 4, e1000088. [Google Scholar] [CrossRef]
- Prentice, E.; McAuliffe, J.; Lu, X.; Subbarao, K.; Denison, M.R. Identification and Characterization of Severe Acute Respiratory Syndrome Coronavirus Replicase Proteins. J. Virol. 2004, 78, 9977–9986. [Google Scholar] [CrossRef]
- Reggiori, F.; Monastyrska, I.; Verheije, M.H.; Calì, T.; Ulasli, M.; Bianchi, S.; Bernasconi, R.; de Haan, C.A.M.; Molinari, M. Coronaviruses Hijack the LC3-I-Positive EDEMosomes, ER-Derived Vesicles Exporting Short-Lived ERAD Regulators, for Replication. Cell Host Microbe 2010, 7, 500–508. [Google Scholar] [CrossRef]
- Shi, S.T.; Schiller, J.J.; Kanjanahaluethai, A.; Baker, S.C.; Oh, J.W.; Lai, M.M. Colocalization and Membrane Association of Murine Hepatitis Virus Gene 1 Products and De Novo-Synthesized Viral RNA in Infected Cells. J. Virol. 1999, 73, 5957–5969. [Google Scholar]
- van der Meer, Y.; Snijder, E.J.; Dobbe, J.C.; Schleich, S.; Denison, M.R.; Spaan, W.J.; Locker, J.K. Localization of Mouse Hepatitis Virus Nonstructural Proteins and RNA Synthesis Indicates a Role for Late Endosomes in Viral Replication. J. Virol. 1999, 73, 7641–7657. [Google Scholar]
- Prentice, E.; Jerome, W.G.; Yoshimori, T.; Mizushima, N.; Denison, M.R. Coronavirus Replication Complex Formation Utilizes Components of Cellular Autophagy. J. Biol. Chem. 2004, 279, 10136–10141. [Google Scholar]
- Zhao, Z.; Thackray, L.B.; Miller, B.C.; Lynn, T.M.; Becker, M.M.; Ward, E.; Mizushima, N.N.; Denison, M.R.; Virgin, H.W., 4th. Coronavirus Replication does Not Require the Autophagy Gene ATG5. Autophagy 2007, 3, 581–585. [Google Scholar]
- Cottam, E.M.; Maier, H.J.; Manifava, M.; Vaux, L.C.; Chandra-Schoenfelder, P.; Gerner, W.; Britton, P.; Ktistakis, N.T.; Wileman, T. Coronavirus nsp6 Proteins Generate Autophagosomes from the Endoplasmic Reticulum via an Omegasome Intermediate. Autophagy 2011, 7, 1335–1347. [Google Scholar] [CrossRef]
- de Haan, C.A.M.; Reggiori, F.; Molinari, M. Autophagy-Independent LC3 Function in Vesicular Traffic. Autophagy 2010, 6, 994–996. [Google Scholar] [CrossRef]
- Stertz, S.; Reichelt, M.; Spiegel, M.; Kuri, T.; Martinez-Sobrido, L.; Garcia-Sastre, A.; Weber, F.; Kochs, G. The Intracellular Sites of Early Replication and Budding of SARS-Coronavirus. Virology 2007, 361, 304–315. [Google Scholar]
- Mizushima, N.; Yamamoto, A.; Matsui, M.; Yoshimori, T.; Ohsumi, Y. In Vivo Analysis of Autophagy in Response to Nutrient Starvation using Transgenic Mice Expressing a Fluorescent Autophagosome Marker. Mol. Biol. Cell 2004, 15, 1101–1111. [Google Scholar]
- Cali, T.; Galli, C.; Olivari, S.; Molinari, M. Segregation and Rapid Turnover of EDEM1 by an Autophagy-Like Mechanism Modulates Standard ERAD and Folding Activities. Biochem. Biophys. Res. Commun. 2008, 371, 405–410. [Google Scholar] [CrossRef]
- Cali, T.; Vanoni, O.; Molinari, M. The Endoplasmic Reticulum Crossroads for Newly Synthesized Polypeptide Chains. Prog. Mol. Biol. Transl. Sci. 2008, 83, 135–179. [Google Scholar] [CrossRef]
- Bernasconi, R.; Galli, C.; Noack, J.; Bianchi, S.; de Haan, C.A.; Reggiori, F.; Molinari, M. Role of the SEL1L:LC3-I Complex as an ERAD Tuning Receptor in the Mammalian ER. Mol. Cell. 2012, 46, 809–819. [Google Scholar] [CrossRef]
- Hagemeijer, M.C.; Ulasli, M.; Vonk, A.; Reggiori, F.; Rottier, P.J.M.; de Haan, C.A.M. Mobility and Interactions of the Coronavirus Nonstructural Protein 4. J. Virol. 2011, 85, 4572–4577. [Google Scholar] [CrossRef]
- Hagemeijer, M.C.; de Haan, C.A.M. Utrecht University: Utrecht, the Netherlands, 2012; Unpublished work.
- Gadlage, M.J.; Sparks, J.S.; Beachboard, D.C.; Cox, R.G.; Doyle, J.D.; Stobart, C.C.; Denison, M.R. Murine Hepatitis Virus Nonstructural Protein 4 Regulates Virus-Induced Membrane Modifications and Replication Complex Function. J. Virol. 2010, 84, 280–290. [Google Scholar] [CrossRef]
- Sparks, J.S.; Lu, X.; Denison, M.R. Genetic Analysis of Murine Hepatitis Virus nsp4 in Virus Replication. J. Virol. 2007, 81, 12554–12563. [Google Scholar] [CrossRef]
- Snijder, E.J.; van Tol, H.; Roos, N.; Pedersen, K.W. Non-Structural Proteins 2 and 3 Interact to Modify Host Cell Membranes during the Formation of the Arterivirus Replication Complex. J. Gen. Virol. 2001, 82, 985–994. [Google Scholar]
- Pedersen, K.W.; van der Meer, Y.; Roos, N.; Snijder, E.J. Open Reading Frame 1a-Encoded Subunits of the Arterivirus Replicase Induce Endoplasmic Reticulum-Derived Double-Membrane Vesicles which Carry the Viral Replication Complex. J. Virol. 1999, 73, 2016–2026. [Google Scholar]
- Posthuma, C.C.; Pedersen, K.W.; Lu, Z.; Joosten, R.G.; Roos, N.; Zevenhoven-Dobbe, J.C.; Snijder, E.J. Formation of the Arterivirus replication/transcription Complex: A Key Role for Nonstructural Protein 3 in the Remodeling of Intracellular Membranes. J. Virol. 2008, 82, 4480–4491. [Google Scholar]
- Miller, S.; Kastner, S.; Krijnse-Locker, J.; Buhler, S.; Bartenschlager, R. The Non-Structural Protein 4A of Dengue Virus is an Integral Membrane Protein Inducing Membrane Alterations in a 2K-Regulated Manner. J. Biol. Chem. 2007, 282, 8873–8882. [Google Scholar] [CrossRef]
- Roosendaal, J.; Westaway, E.G.; Khromykh, A.; Mackenzie, J.M. Regulated Cleavages at the West Nile Virus NS4A-2K-NS4B Junctions Play a Major Role in Rearranging Cytoplasmic Membranes and Golgi Trafficking of the NS4A Protein. J. Virol. 2006, 80, 4623–4632. [Google Scholar]
- Egger, D.; Wolk, B.; Gosert, R.; Bianchi, L.; Blum, H.E.; Moradpour, D.; Bienz, K. Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex. J. Virol. 2002, 76, 5974–5984. [Google Scholar] [CrossRef]
- den Boon, J.A.; Chen, J.; Ahlquist, P. Identification of Sequences in Brome Mosaic Virus Replicase Protein 1a that Mediate Association with Endoplasmic Reticulum Membranes. J. Virol. 2001, 75, 12370–12381. [Google Scholar]
- Echeverri, A.C.; Dasgupta, A. Amino Terminal Regions of Poliovirus 2C Protein Mediate Membrane Binding. Virology 1995, 208, 540–553. [Google Scholar] [CrossRef]
- Lundin, M.; Monne, M.; Widell, A.; von Heijne, G.; Persson, M.A.A. Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B. J. Virol. 2003, 77, 5428–5438. [Google Scholar] [CrossRef]
- Miller, D.J.; Ahlquist, P. Flock House Virus RNA Polymerase is a Transmembrane Protein with Amino-Terminal Sequences Sufficient for Mitochondrial Localization and Membrane Insertion. J. Virol. 2002, 76, 9856–9867. [Google Scholar] [CrossRef]
- Miller, S.; Sparacio, S.; Bartenschlager, R. Subcellular Localization and Membrane Topology of the Dengue Virus Type 2 Non-Structural Protein 4B. J. Biol. Chem. 2006, 281, 8854–8863. [Google Scholar]
- O'Reilly, E.K.; Wang, Z.; French, R.; Kao, C.C. Interactions between the Structural Domains of the RNA Replication Proteins of Plant-Infecting RNA Viruses. J. Virol. 1998, 72, 7160–7169. [Google Scholar]
- O'Reilly, E.; Paul, J.; Kao, C. Analysis of the Interaction of Viral RNA Replication Proteins by using the Yeast Two-Hybrid Assay. J. Virol. 1997, 71, 7526–7532. [Google Scholar]
- Paul, D.; Romero-Brey, I.; Gouttenoire, J.; Stoitsova, S.; Krijnse-Locker, J.; Moradpour, D.; Bartenschlager, R. NS4B Self-Interaction through Conserved C-Terminal Elements is Required for the Establishment of Functional Hepatitis C Virus Replication Complexes. J. Virol. 2011, 85, 6963–6976. [Google Scholar]
- Mackenzie, J.M.; Khromykh, A.A.; Jones, M.K.; Westaway, E.G. Subcellular Localization and some Biochemical Properties of the Flavivirus Kunjin Nonstructural Proteins NS2A and NS4A. Virology 1998, 245, 203–215. [Google Scholar] [CrossRef]
- McMahon, H.T.; Gallop, J.L. Membrane Curvature and Mechanisms of Dynamic Cell Membrane Remodelling. Nature 2005, 438, 590–596. [Google Scholar] [CrossRef]
- Shibata, Y.; Hu, J.; Kozlov, M.M.; Rapoport, T.A. Mechanisms Shaping the Membranes of Cellular Organelles. Annu. Rev. Cell Dev. Biol. 2009, 25, 329–354. [Google Scholar] [CrossRef]
- Zimmerberg, J.; Kozlov, M.M. How Proteins Produce Cellular Membrane Curvature. Nat. Rev. Mol. Cell Biol. 2006, 7, 9–19. [Google Scholar] [CrossRef]
- Jones, D.M.; Gretton, S.N.; McLauchlan, J.; Targett-Adams, P. Mobility Analysis of an NS5A–GFP Fusion Protein in Cells Actively Replicating Hepatitis C Virus Subgenomic RNA. J. Gen. Virol. 2007, 88, 470–475. [Google Scholar]
- Gretton, S.N.; Taylor, A.I.; McLauchlan, J. Mobility of the Hepatitis C Virus NS4B Protein on the Endoplasmic Reticulum Membrane and Membrane-Associated Foci. J. Gen. Virol. 2005, 86, 1415–1421. [Google Scholar]
- Cui, Z.; Zhang, Z.; Zhang, X.; Wen, J.; Zhou, Y.; Xie, W. Visualizing the Dynamic Behavior of Poliovirus Plus-Strand RNA in Living Host Cells. Nucleic Acids Res. 2005, 33, 3245–3252. [Google Scholar] [CrossRef]
- Kujala, P.; Ikaheimonen, A.; Ehsani, N.; Vihinen, H.; Auvinen, P.; Kaariainen, L. Biogenesis of the Semliki Forest Virus RNA Replication Complex. J. Virol. 2001, 75, 3873–3884. [Google Scholar]
- Spuul, P.; Balistreri, G.; Kaariainen, L.; Ahola, T. Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes. J. Virol. 2010, 84, 7543–7557. [Google Scholar] [CrossRef]
- Wolk, B.; Buchele, B.; Moradpour, D.; Rice, C.M. A Dynamic View of Hepatitis C Virus Replication Complexes. J. Virol. 2008, 82, 10519–10531. [Google Scholar] [CrossRef]
- Egger, D.; Bienz, K. Intracellular Location and Translocation of Silent and Active Poliovirus Replication Complexes. J. Gen. Virol. 2005, 86, 707–718. [Google Scholar] [CrossRef]
- Hagemeijer, M.C.; Verheije, M.H.; Ulasli, M.; Shaltiël, I.A.; de Vries, L.A.; Reggiori, F.; Rottier, P.J.M.; de Haan, C.A.M. Dynamics of Coronavirus Replication-Transcription Complexes. J. Virol. 2010, 84, 2134–2149. [Google Scholar]
- Ma, S.; Chisholm, R.L. Cytoplasmic Dynein-Associated Structures Move Bidirectionally in Vivo. J. Cell Sci. 2002, 115, 1453–1460. [Google Scholar]
- Bost, A.G.; Venable, D.; Liu, L.; Heinz, B.A. Cytoskeletal Requirements for Hepatitis C Virus (HCV) RNA Synthesis in the HCV Replicon Cell Culture System. J. Virol. 2003, 77, 4401–4408. [Google Scholar] [CrossRef]
- Hagemeijer, M.C.; Vonk, A.M.; Monastyrska, I.; Rottier, P.J.; de Haan, C.A. Visualizing Coronavirus RNA Synthesis in Time by using Click Chemistry. J. Virol. 2012, 86, 5808–5816. [Google Scholar]
- Imbert, I.; Snijder, E.J.; Dimitrova, M.; Guillemot, J.C.; Lecine, P.; Canard, B. The SARS-Coronavirus PLnc Domain of nsp3 as a replication/transcription Scaffolding Protein. Virus Res. 2008, 133, 136–148. [Google Scholar]
- Pan, J.; Peng, X.; Gao, Y.; Li, Z.; Lu, X.; Chen, Y.; Ishaq, M.; Liu, D.; DeDiego, M.L.; Enjuanes, L.; et al. Genome-Wide Analysis of Protein-Protein Interactions and Involvement of Viral Proteins in SARS-CoV Replication. PLoS One 2008, 3, e3299. [Google Scholar]
- von Brunn, A.; Teepe, C.; Simpson, J.C.; Pepperkok, R.; Friedel, C.C.; Zimmer, R.; Roberts, R.; Baric, R.; Haas, J. Analysis of Intraviral Protein-Protein Interactions of the SARS Coronavirus ORFeome. PLoS One 2007, 2, e459. [Google Scholar]
- Bost, A.G.; Carnahan, R.H.; Lu, X.T.; Denison, M.R. Four Proteins Processed from the Replicase Gene Polyprotein of Mouse Hepatitis Virus Colocalize in the Cell Periphery and Adjacent to Sites of Virion Assembly. J. Virol. 2000, 74, 3379–3387. [Google Scholar]
- Denison, M.R.; Spaan, W.J.; van der Meer, Y.; Gibson, C.A.; Sims, A.C.; Prentice, E.; Lu, X.T. The Putative Helicase of the Coronavirus Mouse Hepatitis Virus is Processed from the Replicase Gene Polyprotein and Localizes in Complexes that are Active in Viral RNA Synthesis. J. Virol. 1999, 73, 6862–6871. [Google Scholar]
- Hurst, K.R.; Ye, R.; Goebel, S.J., Javaraman; Masters, P.S. An Interaction between the Nucleocapsid Protein and a Component of the Replicase-Transcriptase Complex is Crucial for the Infectivity of Coronavirus Genomic RNA. J. Virol. 2010, 84, 10276–10288. [Google Scholar]
- Verheije, M.H.; Hagemeijer, M.C.; Ulasli, M.; Reggiori, F.; Rottier, P.J.M.; Masters, P.S.; de Haan, C.A.M. The Coronavirus Nucleocapsid Protein is Dynamically Associated with the Replication-Transcription Complexes. J. Virol. 2010, 84, 11575–11579. [Google Scholar] [CrossRef]
- Almazan, F.; Galan, C.; Enjuanes, L. The Nucleoprotein is Required for Efficient Coronavirus Genome Replication. J. Virol. 2004, 78, 12683–12688. [Google Scholar] [CrossRef]
- Compton, S.R.; Rogers, D.B.; Holmes, K.V.; Fertsch, D.; Remenick, J.; McGowan, J.J. In Vitro Replication of Mouse Hepatitis Virus Strain A59. J. Virol. 1987, 61, 1814–1820. [Google Scholar]
- Schelle, B.; Karl, N.; Ludewig, B.; Siddell, S.G.; Thiel, V. Selective Replication of Coronavirus Genomes that Express Nucleocapsid Protein. J. Virol. 2005, 79, 6620–6630. [Google Scholar] [CrossRef]
- de Haan, C.A.; Rottier, P.J. Molecular Interactions in the Assembly of Coronaviruses. Adv. Virus Res. 2005, 64, 165–230. [Google Scholar] [CrossRef]
- van Hemert, M.J.; van den Worm, S.H.; Knoops, K.; Mommaas, A.M.; Gorbalenya, A.E.; Snijder, E.J. SARS-Coronavirus replication/transcription Complexes are Membrane-Protected and Need a Host Factor for Activity in Vitro. PLoS Pathog. 2008, 4, e1000054. [Google Scholar]
- Schwartz, M.; Chen, J.; Janda, M.; Sullivan, M.; den Boon, J.; Ahlquist, P. A Positive-Strand RNA Virus Replication Complex Parallels Form and Function of Retrovirus Capsids. Mol. Cell 2002, 9, 505–514. [Google Scholar] [CrossRef]
- Kopek, B.G.; Perkins, G.; Miller, D.J.; Ellisman, M.H.; Ahlquist, P. Three-Dimensional Analysis of a Viral RNA Replication Complex Reveals a Virus-Induced Mini-Organelle. PLoS Biol. 2007, 5, e220. [Google Scholar]
- Westaway, E.G.; Khromykh, A.A.; Mackenzie, J.M. Nascent Flavivirus RNA Colocalizedin Situwith Double-Stranded RNA in Stable Replication Complexes. Virology 1999, 258, 108–117. [Google Scholar] [CrossRef]
- Gillespie, L.K.; Hoenen, A.; Morgan, G.; Mackenzie, J.M. The Endoplasmic Reticulum Provides the Membrane Platform for Biogenesis of the Flavivirus Replication Complex. J. Virol. 2010, 84, 10438–10447. [Google Scholar] [CrossRef]
- Welsch, S.; Miller, S.; Romero-Brey, I.; Merz, A.; Bleck, C.K.; Walther, P.; Fuller, S.D.; Antony, C.; Krijnse-Locker, J.; Bartenschlager, R. Composition and Three-Dimensional Architecture of the Dengue Virus Replication and Assembly Sites. Cell Host Microbe 2009, 5, 365–375. [Google Scholar] [CrossRef]
- Sola, I.; Mateos-Gomez, P.A.; Almazan, F.; Zuniga, S.; Enjuanes, L. RNA-RNA and RNA-Protein Interactions in Coronavirus Replication and Transcription. RNA Biol. 2011, 8, 237–248. [Google Scholar] [CrossRef]
- Diaz, A.; Wang, X.; Ahlquist, P. Membrane-Shaping Host Reticulon Proteins Play Crucial Roles in Viral RNA Replication Compartment Formation and Function. Proc. Natl. Acad. Sci. USA 2010, 107, 16291–16296. [Google Scholar] [CrossRef]
- Hu, J.; Shibata, Y.; Zhu, P.P.; Voss, C.; Rismanchi, N.; Prinz, W.A.; Rapoport, T.A.; Blackstone, C. A Class of Dynamin-Like GTPases Involved in the Generation of the Tubular ER Network. Cell 2009, 138, 549–561. [Google Scholar]
- Hu, J.; Shibata, Y.; Voss, C.; Shemesh, T.; Li, Z.; Coughlin, M.; Kozlov, M.M.; Rapoport, T.A.; Prinz, W.A. Membrane Proteins of the Endoplasmic Reticulum Induce High-Curvature Tubules. Science 2008, 319, 1247–1250. [Google Scholar] [CrossRef]
- Voeltz, G.K.; Prinz, W.A.; Shibata, Y.; Rist, J.M.; Rapoport, T.A. A Class of Membrane Proteins Shaping the Tubular Endoplasmic Reticulum. Cell 2006, 124, 573–586. [Google Scholar]
- Klopfenstein, D.R.; Klumperman, J.; Lustig, A.; Kammerer, R.A.; Oorschot, V.; Hauri, H. Subdomain-Specific Localization of Climp-63 (P63) in the Endoplasmic Reticulum is Mediated by its Luminal α-Helical Segment. J. Cell Biol. 2001, 153, 1287–1300. [Google Scholar] [CrossRef]
- Farsad, K.; Camilli, P.D. Mechanisms of Membrane Deformation. Curr. Opin. Cell Biol. 2003, 15, 372–381. [Google Scholar] [CrossRef]
- Shibata, Y.; Shemesh, T.; Prinz, W.A.; Palazzo, A.F.; Kozlov, M.M.; Rapoport, T.A. Mechanisms Determining the Morphology of the Peripheral ER. Cell 2010, 143, 774–788. [Google Scholar] [CrossRef]
- Hsu, N.; Ilnytska, O.; Belov, G.; Santiana, M.; Chen, Y.; Takvorian, P.M.; Pau, C.; van der Schaar, H.; Kaushik-Basu, N.; Balla, T.; et al. Viral Reorganization of the Secretory Pathway Generates Distinct Organelles for RNA Replication. Cell 2010, 141, 799–811. [Google Scholar]
- Reiss, S.; Rebhan, I.; Backes, P.; Romero-Brey, I.; Erfle, H.; Matula, P.; Kaderali, L.; Poenisch, M.; Blankenburg, H.; Hiet, M.; et al. Recruitment and Activation of a Lipid Kinase by Hepatitis C Virus NS5A is Essential for Integrity of the Membranous Replication Compartment. Cell Host Microbe 2011, 9, 32–45. [Google Scholar] [CrossRef]
- Guinea, R.; Carrasco, L. Phospholipid Biosynthesis and Poliovirus Genome Replication, Two Coupled Phenomena. EMBO J. 1990, 9, 2011–2016. [Google Scholar]
- Heaton, N.S.; Perera, R.; Berger, K.L.; Khadka, S.; LaCount, D.J.; Kuhn, R.J.; Randall, G. Dengue Virus Nonstructural Protein 3 Redistributes Fatty Acid Synthase to Sites of Viral Replication and Increases Cellular Fatty Acid Synthesis. Proc. Natl. Acad. Sci. USA 2010, 107, 17345–17350. [Google Scholar]
- Perez, L.; Guinea, R.; Carrasco, L. Synthesis of Semliki Forest Virus RNA Requires Continuous Lipid Synthesis. Virology 1991, 183, 74–82. [Google Scholar]
- Perera, R.; Riley, C.; Isaac, G.; Hopf-Jannasch, A.S.; Moore, R.J.; Weitz, K.W.; Pasa-Tolic, L.; Metz, T.O.; Adamec, J.; Kuhn, R.J. Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells. PLoS Pathog. 2012, 8, e1002584. [Google Scholar] [CrossRef]
- Lippincott-Schwartz, J.; Altan-Bonnet, N.; Patterson, G.H. Photobleaching and Photoactivation: Following Protein Dynamics in Living Cells. Nat. Cell. Biol. Suppl., S7–S14.
- Lippincott-Schwartz, J.; Patterson, G.H. Development and use of Fluorescent Protein Markers in Living Cells. Science 2003, 300, 87–91. [Google Scholar] [CrossRef]
- Fusco, D.; Accornero, N.; Lavoie, B.; Shenoy, S.M.; Blanchard, J.; Singer, R.H.; Bertrand, E. Single mRNA Molecules Demonstrate Probabilistic Movement in Living Mammalian Cells. Curr. Biol. 2003, 13, 161–167. [Google Scholar] [CrossRef]
- Daigle, N.; Ellenberg, J. LambdaN-GFP: An RNA Reporter System for Live-Cell Imaging. Nat. Methods 2007, 4, 633–636. [Google Scholar] [CrossRef]
- Tyagi, S.; Kramer, F.R. Molecular Beacons: Probes that Fluoresce upon Hybridization. Nat. Biotechnol. 1996, 14, 303–308. [Google Scholar] [CrossRef]
- Baskin, J.M.; Prescher, J.A.; Laughlin, S.T.; Agard, N.J.; Chang, P.V.; Miller, I.A.; Lo, A.; Codelli, J.A.; Bertozzi, C.R. Copper-Free Click Chemistry for Dynamic in Vivo Imaging. Proc. Natl. Acad. Sci. USA 2007, 104, 16793–16797. [Google Scholar]
- Betzig, E.; Patterson, G.H.; Sougrat, R.; Lindwasser, O.W.; Olenych, S.; Bonifacino, J.S.; Davidson, M.W.; Lippincott-Schwartz, J.; Hess, H.F. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 2006, 313, 1642–1645. [Google Scholar] [CrossRef]
- Rust, M.J.; Bates, M.; Zhuang, X. Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy (STORM). Nat. Methods 2006, 3, 793–795. [Google Scholar] [CrossRef]
- Hell, S.W.; Wichmann, J. Breaking the Diffraction Resolution Limit by Stimulated Emission: Stimulated-Emission-Depletion Fluorescence Microscopy. Opt. Lett. 1994, 19, 780–782. [Google Scholar]
- van Rijnsoever, C.; Oorschot, V.; Klumperman, J. Correlative Light-Electron Microscopy (CLEM) Combining Live-Cell Imaging and Immunolabeling of Ultrathin Cryosections. Nat. Methods 2008, 5, 973–980. [Google Scholar] [CrossRef]
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).