SARS Coronavirus Replication Is Supported by a Reticulovesicular Network of Modified Endoplasmic Reticulum Ke vin Knoops 1 2 Marjolein Kikkert 2 Sjoerd H E van den Worm 2 Jessika C Zevenhoven Dobbe 2 Yvonne van der Meer 2 Abraham J Koster 1 A Mieke Mommaas 1 Eric J Snijder 2 1 Section Electron Microscopy Department of Molecular Cell Biology Leiden University Medical Center Leiden The Netherlands 2 Molecular Virology Laboratory Department of Medical Microbiology Leiden University Medical Center Leiden The Netherlands Positive strand RNA viruses a large group including human pathogens such as SARS coronavirus SARS CoV replicate in the cytoplasm of infected host cells Their replication complexes are commonly associated with modified host cell membranes Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles Generally their ultrastructure morpho genesis and exact role in viral replication remain to be defined Poorly characterized double membrane vesicles DMVs were previously implicated in SARS CoV RNA synthesis We have now applied electron tomography of cryofixed infected cells for the three dimensional imaging of coronavirus induced membrane alterations at high resolution Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes numerous interconnected DMVs diameter 200 300 nm and vesicle packets apparently arising from DMV merger The convoluted membranes were most abundantly immunolabeled for viral replicase subunits However double stranded RNA presumably revealing the site of viral RNA synthesis mainly localized to the DMV interior Since we could not discern a connection between DMV interior and cytosol our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS CoV RNA synthesis Our data document the extensive virus induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms Together with biochemical studies of the viral enzyme complex our ultrastructural description of this replication network will aid to further dissect the early stages of the coronavirus life cycle and its virus host interactions Citation Knoops K Kikkert M van den Worm SHE Zevenhoven Dobbe JC van der Meer Y et al 2008 SARS coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum PLoS Biol 6 9 e226 doi 10 1371 journal pbio 0060226 Introduction Viruses rely on the host cell s infrastructure and metabo lism during essentially all stages of their replication cycle and have therefore adopted strategies to coordinate a variety of molecular interactions in both time and intracellular space The fact that the replication complexes of positive strand RNA RNA viruses of eukaryotes are invariably associated with modified intracellular membranes appears to be a striking example of such a strategy 1 8 Specific RNA virus replicase subunits are targeted to the membranes of particular cell organelles that are subsequently modified into characteristic structures with which viral RNA synthesis is associated The morphogenesis ultrastructure and function of these complexes sometimes referred to as viral factories are only beginning to be understood They may facilitate the concentration of viral macromolecules and provide a membrane based structural framework for RNA synthesis Other potential benefits include the possibility to coordinate different steps in the viral life cycle and to delay the induction of host defense mechanisms that can be triggered by the double stranded RNA dsRNA intermediates of RNA virus replication 2 9 10 Defining the structure function relationships that govern the membrane associated replica tion of RNA viruses a large virus cluster including many important pathogens will enhance our general understand ing of their molecular biology and may have important implications for the development of novel antiviral control strategies Following the 2003 outbreak of severe acute respiratory syndrome SARS for a review see 11 the coronavirus family of RNA viruses received worldwide attention In addition to SARS coronavirus SARS CoV several other novel family members were identified including two that also infect humans 12 Coronaviruses and other members of the nidovirus group have a polycistronic genome and employ Academic Editor Michael Emerman Fred Hutchinson Cancer Research Center United States of America Received March 4 2008 Accepted August 4 2008 Published September 16 2008 Copyright C211 2008 Knoops et al This is an open access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited Abbreviations 3 D three dimensional CM convoluted membranes DMV double membrane vesicle dsRNA double stranded RNA EM electron microscopy ER endoplasmic reticulum ERGIC endoplasmic reticulum Golgi intermediate com partment ET electron tomography FS freeze substitution IEM immunoelectron microscopy IF immunofluorescence h p i hours postinfection MHV mouse hepatitis virus nsp nonstructural protein ORF open reading frame RdRp RNA depended RNA polymerase RNA positive strand RNA RTC replication tran scription complex SARS severe acute respiratory syndrome SARS CoV severe acute respiratory syndrome coronavirus VP vesicle packet To whom correspondence should be addressed E mail e j snijder lumc nl PLoS Biology www plosbiology org September 2008 Volume 6 Issue 9 e2261957 PL o SBIOLOGY various transcriptional and post translational mechanisms to regulate its expression 13 14 The gene encoding the replicase transcriptase commonly referred to as replicase comprises about two thirds of the coronavirus genome which at 27 31 kb is the largest RNA genome known to date The replicase gene consists of open reading frames ORFs 1a and 1b of which the latter is expressed by a ribosomal frameshift near the 39 end of ORF1a Thus SARS CoV genome translation yields two polyproteins pp1a and pp1ab that are autoproteolytically cleaved into 16 non structural proteins nsp1 to 16 Figure 1 by proteases residing in nsp3 and nsp5 15 17 Several of the replicative enzymes of coronaviruses like an RNA dependent RNA polymerase RdRp and a helicase are common among RNA viruses but they also contain a variety of functions that are rare or absent in other RNA viruses including a set of intriguing proteins that are distantly related to cellular RNA processing enzymes 13 14 18 The complexity of coronavirus RNA synthesis is further highlighted by the fact that it entails not only the production of new genome molecules from full length negative strand RNA replication but also a unique mechanism of discontinuous RNA synthesis to generate subgenome length negative strand RNA templates for sub genomic mRNA production transcription 19 20 The resulting set of subgenomic transcripts eight in the case of SARS CoV serves to express structural and accessory protein genes in the 39 proximal domain of the genome Ultimately new coronavirions are assembled by budding of nucleocap sids into the lumen of pre Golgi membrane compartments 21 22 The nidovirus replicase includes several presumed multi spanning transmembrane proteins that are thought to physi cally anchor the replication transcription complex RTC to intracellular membranes In the case of coronaviruses these domains reside in nsp3 nsp4 and nsp6 Figure 1 23 24 In the cytoplasm of infected cells nidoviruses induce the formation of typical paired membranes and double mem brane structures that have commonly been referred to as double membrane vesicles DMVs 25 28 These struc tures are mainly found in the perinuclear area of the cell where according to immunofluorescence IF microscopy studies de novo made viral RNA and various replicase subunits colocalize presumably in the viral RTC 16 17 28 29 Immunoelectron microscopy IEM previously revealed that SARS CoV nsp3 and nsp13 localize to the outside of DMVs and or the region between DMVs Although these proteins also colocalized in part with endoplasmic reticulum ER marker proteins 26 28 30 the origin of DMV membranes has remained undecided since other studies have implicated other organelles in the formation of RTCs and DMVs e g late endosomes autophagosomes and most recently the early secretory pathway and potentially also mitochondria 31 35 Previous ultrastructural studies may have been hampered by the technical challenge of DMV preservation 28 In particular the DMV inner structure is fragile and loss or collapse of DMV contents likely was a complicatingfactor Althoughtheuseofcryofixation methods dramatically improved DMV preservation 28 our under standing of the three dimensional 3 D organization and origin of DMVs was hampered by the inherent limitations of analyzing conventional thin sections 100 nm by electron microscopy EM in particular since the diameter of DMVs was estimated to be between 200 and 350 nm 28 To develop a 3 D ultrastructural model for the RTC related Figure 1 The Coronavirus Replicase Polyprotein The domain organization and proteolytic processing map of the SARS CoV replicase polyprotein pp1ab The replicase cleavage products nsp1 16 are numbered and conserved domains are highlighted blue conserved across nidoviruses grey conserved in coronaviruses These include transmembrane domains TM protease domains PLP and MP and putative RNA primase P helicase HEL exonuclease Exo endoribonuclease N and methyl transferase MT activities For more details see 14 18 The delineation of amino acids encoded in ORF1a and ORF1b is indicated as RFS ribosomal frameshift and arrows represent sites in pp1ab that are cleaved by the nsp3 papain like protease in blue or the nsp5 main protease in red doi 10 1371 journal pbio 0060226 g001 PLoS Biology www plosbiology org September 2008 Volume 6 Issue 9 e2261958 SARS CoV Replication Complex Author Summary Viruses with a positive stranded RNA genome replicate in the cytoplasm of infected host cells Their replication is driven by a membrane bound viral enzyme complex that is commonly asso ciated with modified intracellular membranes Little is understood about the formation and architecture of these replication structures and their exact role in viral RNA synthesis We used electron microscopy and tomography for the three dimensional imaging of the membrane alterations induced by severe acute respiratory syndrome SARS coronavirus a member of the virus group with the largest RNA genome known to date Previously coronaviruses were reported to induce large numbers of isolated double membrane vesicles DMVs However our present studies reveal an elaborate reticulovesicular network of modified endoplasmic reticulum membranes with which SARS coronavirus replicative proteins are associated The lumen of this unique membrane network contains numerous large diameter 250 300 nm inner vesicles which were formerly thought to reside in isolated DMVs Intriguingly although the interior of these vesicles does not appear to be connected to the cytosol it labels abundantly for double stranded RNA which presumably is present at the site of viral RNA synthesis The ultrastructural dissection of this elaborate replication network shows how coronaviruses extensively reorganize the host cell s membrane infrastructure to coordinate their replication cycle and possibly also hide replicating RNA from antiviral defense mecha nisms membrane alterations in SARS CoV infected cells we have now employed electron tomography ET for reviews see 36 37 This technique uses a set of two dimensional 2 D transmission EM images recorded at different specimen tilt angles with respect to the primary beam for calculating a 3 D image tomogram Typically the specimen is tilted over a range of 6658 in small tilt increments 18 and an image is recorded at each tilt angle The tomograms of infected cells allowed us to trace DMV membranes and establish previously unnoticed structural connections In particular ET revealed that coronavirus DMVs are not isolated vesicles but instead are integrated into a unique reticulovesicular network of modified ER membranes which also includes convoluted membranes that were not previously implicated in viral RNA synthesis Strikingly the latter structure and not the DMVs were primarily immunolabeled using antibodies recognizing viral replicase subunits In contrast immunolab eling with an antibody recognizing presumably viral dsRNA abundantly labeled the DMV interior Since we could not discern a connection between the DMV interior and cytosol our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS CoV RNA synthesis The virus induced replication network docu mented here places the early stages of the viral lifecycle and accompanying virus host interactions in a new perspective Results SARS Coronavirus Infection Induces Multiple Distinct Membrane Alterations Previously we experienced that compared to more tradi tional chemical fixation protocols the preservation of the fragile coronavirus DMV structures could be significantly improved by using a combination of cryofixation and freeze substitution FS 28 We now further refined the FS protocol in particular by improving membrane contrast by adding 10 water to the FS medium 38 Using these optimized conditions to prepare thin sections 100 nm of SARS CoV infected Vero E6 cells we could detect the first DMVs at 2 h postinfection h p i and were able to monitor the subsequent development of virus induced membrane alterations Early DMVs had sizes ranging from 150 to 300 nm were distributed throughout the cytoplasm and were sometimes located in the proximity of small reticular membranes with which occasionally they appeared to be connected Figure 2A From 4 h p i on the number of DMVs increased dramatically and DMV clusters were observed throughout the cell again frequently accom panied by and sometimes clearly connected to reticular membrane structures Figure 2B arrow As infection pro gressed DMVs became increasingly concentrated in the perinuclear area of the cell Figure 2C in accordance with the available IF microscopy data for various SARS CoV replicase subunits 16 28 29 At 7 h p i a 100 nm thick slice through the center of an infected Vero E6 cell generally contained between 200 and 300 DMVs Initially the DMV inner and outer membranes were generally tightly apposed but occasionally some luminal space between the two lipid bilayers could be discerned Figure 2B arrowhead Although similar observations were previously made for different nidoviruses using a variety of chemical and cryofixation protocols and despite the generally excellent preservation of cellular membranes the documented fragility of coronavirus DMVs makes it clear that we cannot formally exclude the possibility that these local separations could result from preparation damage From 3 h p i on we also observed large assemblies of convoluted membranes CM often in close proximity to DMV clusters Figure 2D These structures with diameters ranging from 0 2 to 2 lm are probably identical to the reticular inclusions that were first observed in cells infected with mouse hepatitis coronavirus MHV more than 40 y ago 39 and were later referred to as clusters of tubular cisternal elements which may have a connection to the ER Golgi intermediate compartment ERGIC 21 We noticed that the SARS CoV induced CM resembled one of the replication related membrane alterations induced by flaviviruses which were proposed to be the site of viral genome translation and polyprotein processing 3 40 41 In some of our images the SARS CoV induced CM appeared to be continuous with both DMV outer membranes Figure 2D inset and ER cisternae suggesting a link to the viral RTC also in coronaviruses Especially at later stages of SARS CoV infection generally beyond 7 h p i we observed packets of single membrane vesicles surrounded by a common outer membrane as previously described by Goldsmith et al 27 The diameter of these vesicle packets VPs ranged from 1 to 5 lm and they sometimes included more than 25 inner vesicles Figure 2E In terms of size morphology electron density and immuno labeling properties see below the vesicles contained in VPs strongly resembled the inner vesicles of DMVs as seen at earlier time points During these later stages of infection the clustered single DMVs Figure 2C gradually disappeared suggesting their merger into the VPs The average outer diameter of DMV inner vesicles at 4 h p i was 250 6 50 nm n 99 whereas later in infection their average diameter DMVs and VPs combined increased to about 300 nm 310 6 50 nm at 7 h p i 300 6 50 lm at 10 h p i Our observations define VPs as a third distinct modifica tion of intracellular membranes that is induced by SARS CoV infection By 10 h p i VPs appeared to have merged into even larger cytoplasmic vacuoles containing both vesicles as well as significant numbers of budding and completed virions Figure 2E DMVs CM and VPs were not observed in mock infected Vero E6 cells Electron Tomography Reveals a Reticulovesicular Network of Modified ER Membranes in SARS CoV Infected Cells Although occasionally the analysis of conventional thin sections suggested CM and DMV outer membranes to be continuous and connected to ER cisternae a more accurate assessment required an analysis in three dimensions We therefore employed ET of semi thick 200 nm sections of cryofixed SARS CoV infected Vero E6 cells By using a specimen holder that could also be tilted around a second axis perpendicular tobothelectron beamand firsttiltaxis we obtained datasets each consisting of 262 differently tilted 2 D images which were used to produce a high resolution reconstruction in three dimensions Such dual axis tomo grams allowed us to visualize and analyze membrane continuities between the respective structures defined in the previous paragraph as illustrated by Videos S1 S4 and Figures 3 5 The analysis was performed at 7 h p i a time point at which the various membrane alterations were all PLoS Biology www plosbiology org September 2008 Volume 6 Issue 9 e2261959 SARS CoV Replication Complex Figure 2 Overview of Membrane Structures Induced by SARS CoV Infection Electron micrographs of SARS CoV infected Vero E6 cells The cells were cryofixed and freeze substituted at 2 h p i A 8 h p i B D or 10 h p i E A Early DMV as observed in a few sections showing a connection arrow to a reticular membrane PLoS Biology www plosbiology org September 2008 Volume 6 Issue 9 e2261960 SARS CoV Replication Complex abundantly present in the absence of advanced cytopathology Nevertheless in some cells infection had progressed more thaninothers allowingthevisualizationofbothadvancedand earlier stages of infection in the same specimen Two major conclusions from this ET analysis were 1 that most or likely all coronavirus DMVs are interconnected by their outer membrane and 2 that they are part of an elaborate network that is continuous with the rough ER As illustrated by the 3 D reconstruction in Figure 3 for most DMVs we observed one or multiple thin 8 nm in diameter neck like connections of their outer membrane to the outer membranes of other DMVs to CM and to cisternae of the rough ER Figure 3 insets For example in the two tomograms used for Videos S1 and S3 at least one such connection was visible for 77 out of 81 DMVs analyzed strongly suggesting that for the remaining DMVs such outer membrane connections existed but fell outside the volume reconstructed using these particular tomograms Of the 77 DMVs for which at least one outer membrane connection was detected 38 had a single connection whereas 27 nine and three DMVs had two three and four connections respec tively Of these 131 co