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CX Reggiori 2008 Gosert et al 2002 Miller and Krijnse Locker 2008 Salonen et al 2005 A recent analysis of SARS CoV and MHV infected cells by electron tomography has revealed therefore the activity of the ERAD machinery must be main tained low to avoid premature interruption of folding programs and favor attainment of the native structure over degradation that these DMVs are part of a reticular network of modified endoplasmic reticulum ER membranes and contain double stranded RNA dsRNA in their interior Knoops et al 2008 of immature polypeptides Cal et al 2008a Consistently it has been reported that EDEM1 a crucial regulator of ERAD Molinari et al 2003 Oda et al 2003 is selectively cleared lation of EDEM1 and OS 9 another short living ER chaperone in the DMVs DMVs are coated with the nonlipidatedLC3 Atg8autophagymarker Downregu lation of LC3 but not inactivation of host cell autoph agy protects cells from CoV infection Our study identifies the host cellular pathway hijacked for supplying CoV replication membranes and describes anautophagy independentrolefornonlipidatedLC3 I INTRODUCTION As an early and crucial event upon infection coronaviruses CoV such as the severe acute respiratory syndrome SARS CoV and mouse hepatitis virus MHV induce the formation of double membrane vesicles DMVs in host cells and target their RTCs on the limiting membranes of these structures de Haan and essential for DMV formation remain mysterious Knoops et al 2008 The possible involvement of the autophagy machinery in the conversion of host membranes into DMVs has been reported However whereas Atg5 an essential component of the autophagy machinery Mizushima et al 2001 Yoshimori and Noda 2008 has been shown to be dispensable for MHV replication Zhao et al 2007 contradictory immunofluores cence IF data report the presence Prentice et al 2004 Zhao et al 2007 or the absence de Haan and Reggiori 2008 Snijder et al 2006 of the autophagosome protein marker LC3 Atg8 on DMVs de Haan and Reggiori 2008 In this study we have used MHV the virus prototype for the CoV biology investigation to unveil the origin of the virus induced DMVs and to identify host cell factors essential for CoV replication In the ER newly synthesized unstructured polypeptides can attract the folding as well as the degradation machineries that are operating in the lumen of this organelle Cal et al 2008b Hebert and Molinari 2007 Under normal growth conditions Coronaviruses Hijack the LC3 I Positive EDEMosomes ER Derived Short Lived ERAD Regulators Fulvio Reggiori 1 6 Iryna Monastyrska 1 6 Monique H Verheije Riccardo Bernasconi 3 Cornelis A M de Haan 2 and Maurizio 1 Department of Cell Biology University Medical Centre Utrecht 3584 2 Virology Division Department of Infectious Diseases and Immunology 3 Institute for Research in Biomedicine 6500 Bellinzona Switzerland 4 Department of Biochemistry University of Padova 35122 Padova Italy 5 Ecole Polytechnique Fe de rale de Lausanne School of Life Sciences 6 These authors contributed equally to this work Correspondence f reggiori umcutrecht nl F R c a m dehaan uu nl DOI 10 1016 j chom 2010 05 013 SUMMARY Coronaviruses CoV including SARS and mouse hepatitis virus MHV are enveloped RNA viruses that induce formation of double membrane vesicles DMVs and target their replication and transcription complexes RTCs on the DMV limiting membranes The DMV biogenesis has been connected with the early secretory pathway CoV induced DMVs how ever lack conventional endoplasmic reticulum ER or Golgi protein markers leaving their membrane origins in question We show that MHV co opts the host cell machinery for COPII independent vesicular ER export of a short living regulator of ER associated degradation ERAD EDEM1 to derive cellular mem branes for replication MHV infection causes accumu 500 Cell Host Kanjanahaluethai et al 2007 Oostra et al 2008 Moreover nsp4 localizes to the ER when separately expressed and moves to the DMVs upon viral infection Oostra et al 2007 Whereas previous work has shown that the early secretory pathway and CoV induced DMV biogenesis are closely connected Knoops et al 2010 Oostra et al 2007 Ver heije et al 2008 the lack of ER ER Golgi intermediate compart ment ERGIC or Golgi protein markers in CoV induced DMVs suggests that their biogenesis does not depend on the conven tional routing of proteins through this transport pathway Oostra et al 2007 Snijder et al 2006 Verheije et al 2008 Thus even though CoV must hijack ER derived host cell membranes for replication the precise origin of the DMV lipid bilayers the host protein content and the identity of the cellular factors from the ER to tune down the ERAD activity Cal et al 2008a This regulation mechanism has been named ERAD tuning It relies on the selective sorting of EDEM1 and probably other short living ER chaperones in 200 800 nm vesicles called EDE Mosomes These vesicles coated with nonlipidated LC3 Atg8 LC3 I Cal et al 2008a 2008b emerge from the ER through a COPII coat independent mechanism Zuber et al 2007 and deliver their content to endosomal compartments for disposal in a series of poorly characterized events Cal et al 2008a 2008b Le Fourn et al 2009 Zuber et al 2007 We have discovered that MHV exploits the pathway of EDE Mosome formation to generate the DMVs required for viral repli cation In doing so MHV interferes with the degradation of EDEM1 and OS 9 another short living chaperone that we iden tify here as a second EDEMosome cargo by trapping them into the DMVs Our data are consistent with the model in which DMVs are generated from the host ER Knoops et al 2008 and explain the absence of conventional ER resident chaperones in their interior In addition we show that whereas the autophagy pathway is not essential for MHV infection nonlipidated LC3 I coats CoV induced DMVs and is essential for MHV replication RESULTS AND DISCUSSION MHV Infection Does Not Require an Intact Autophagy Machinery To conclusively establish whether autophagy is required for MHV infection we assessed the consequences of deleting ATG7 a gene essential for autophagy Komatsu et al 2005 on DMV biogenesis IF analysis of two RTC components nsp2 and nsp3 in MHV infected wild type Atg7 and ATG7 knockout mouse embryonic fibroblasts Atg7 C0 C0 MEF revealed that these two proteins were similarly distributed to numerous punctuate structures in both cell lines Figure 1A These puncta represent the virus induced DMVs that contain viral dsRNA colocalization in Figure 1A Harcourt et al 2004 Knoops et al 2008 The presence in both Atg7 and Atg7 C0 C0 MEF of these double membrane structures with a diameter of approximately 200 350 nm Knoops et al 2008 Stertz et al 2007 was confirmed by conventional electron microscopy Figure 1B Moreover cells with and withoutATG7were equally susceptible to MHV infection This was established by assessing the virus replication using a re combinant luciferase expressing MHV Figure 1C and by deter mining the titer of a virus stock on these cells by using the mean tissue culture infection dose TCID 50 test Figure 1D MHV repli cation in Atg7 and Atg7 C0 C0 MEF during the course of an infection was also very similar Figure 1E Thus the conventional host cell autophagy is not required for formation of MHV induced DMVs nor for viral replication and production of viral progeny Nonlipidated LC3 Atg8 Associates with CoV Induced DMVs Because contrasting data have been published on the presence Cell Host Zhao et al 2007 or the absence de Haan and Reggiori 2008 Snijder et al 2006 of LC3 Atg8 on CoV induced DMVs we examined this issue Our analysis by IF showed that endogenous LC3 extensively colocalized with the DMV protein markers nsp2 and nsp3 Figures 2A and 2C This colocalization was observed during the entire course of the Cell Host MHV infection Figures S1A and S1B available online In contrast ectopically expressed GFP LC3 a conventional protein marker for autophagosome membranes Klionsky et al 2008 Mizushima et al 2004 did not colocalize with nsp2 and nsp3 Figures 2B and 2C These apparently conflicting data were confirmed in other cell lines e g in HeLa cells Figures S1C and S1D and explain the contradictory data in the literature LC3 is present in the cell predominantly in a cytoplasmic form LC3 I that upon autophagy induction is converted into an active lipidated form LC3 II by specific covalent linkage to the phos phatidylethanolamine present on autophagosomal membranes Klionsky et al 2008 Mizushima et al 2004 The lipidation of LC3 I and the formation of LC3 II coated autophagosomes require several proteins including Atg7 Komatsu et al 2005 However Atg7 was not necessary for the association of endoge nous LC3 to DMVs Figure 2D and 2E Consequently Atg7 and LC3 lipidation are not essential for the formation of LC3 positive DMVs Analysis of the protein content in DMVs induced upon MHV nsp2GFP infection of HeLa cells and separated on contin uous density gradients as described Cal et al 2008a showed the presence of LC3 I in the denser fractions containing the DMV protein marker nsp2 GFP Figure 2F These fractions were clearly separated from the lighter autophagosomes contain ing LC3 II that floated at the top of the gradient Moreover IF anal yses revealed that ectopically expressed C terminally HA tagged nonlipidable LC3 localizes on DMVs Figure 2G Taken all together these data show that an intact host autophagy machinery is dispensable for the virus life cycle and that lipidation is not required for LC3 association with the DMV membranes Analogies between MHV Induced DMVs and EDEMosomes The ER origin of the MHV induced DMVs Knoops et al 2008 Oostra et al 2007 the absence of conventional ER markers in their membranes and lumen Oostra et al 2007 Snijder et al 2006 Verheije et al 2008 their association with LC3 I and the fact that DMVs are stained with antibodies against endoge nous LC3 but not with ectopically expressed GFP LC3 Figures 2 S1C and S1D are features reminiscent of those describing the EDEMosomes Cal et al 2008a Significantly the LC3 I coat distinguishes DMVs this study and EDEMosomes Cal et al 2008a Figure S3D from autophagosomes which are associated with LC3 II and can be decorated with GFP LC3 Klionsky et al 2008 Mizushima et al 2004 As in the case of formation of the ER derived CoV induced DMVs it is unclear whether an active autophagy machinery is required for formation of ER derived EDEMosomes and or for disposal of EDEM1 Cal et al 2008a versus Le Fourn et al 2009 To better understand this we compared variations in the intracellular levels of EDEM1 and of p62 a canonical substrate of autophagy Bj rk y et al 2005 under conditions that either inac tivate e g ATG7 deletion Figures 3A and 3B or cell incubation with chloroquine CQ Figures 3Cand3D Klionsky et al 2008 Komatsu et al 2005 or activate autophagy e g rapamycin treat ment Figures 3Cand3D Klionsky et al 2008 Deletion of ATG7 inhibits the p62 turnover Waguri and Komatsu 2009 thus substantially increasing the intracellular level of this autophagy substrate Figure 3A On the contrary deletion of ATG7 did not result in substantial variations of the level of EDEM1 Figure 3A Bj rk y et al 2009 CQ de layed EDEM1 turnover and resulted in intracellular accumulation of EDEM1 Figures 3C and 3D Cal et al 2008a Finally induction of autophagy with rapa mycin reduced the intracellular levels of p62 as expected for an autophagy substrate Figure 3C Bj rk y et al 2009 but increased those of EDEM1 Figure 3C by delaying its turnover Figure 3D These results confirmed that as reported above for CoV replication the pathway regulating EDEM1 turnover is clearly distinct from autophagy They also highlight another analogy between the ERAD tuning pathway and the CoV infection Cell expo sure to the autophagy inducer rapamycin negatively affected both EDEM1 turnover Figures 3C and 3D and MHV replication g the levels of both the N nucleocapsid cells infected with the recombinant lucif Figure S2 All together these observa esize that MHV hijacks the ERAD tuning llular membranes for DMV generation A endogenous LC3 nsp2 nsp3 merged inset Cell Host Zuber et al 2007 CoV induced DMVs and EDEMosomes do not contain conventional ER chaperones or protein markers of the early compartments of the secretory pathway Cal et al 2008a Oostra et al 2007 Snijder et al 2006 Stertz et al B D FG GFP LC3 nsp2 nsp3 merged inset endogenous LC3 nsp2 nsp3 merged inset Atg7 Atg7 23456789101 LC3 I DMVsautophagosomes LC3 II LC3 HA nsp2 GFP LC3 I EDEM1 Figure 2 Autophagy Independent Recruitment of LC3 I onto MHV Induced A and B HEK293 cells stably transfected B or not A with a plasmid expressing C Summary statistics of the samples shown in A and B expressed as the percenta bars represent the standard error of the mean percentage from counting 40 cells are significantly different t df 78 10 4 p 0 00001 D Atg7 and Atg7 C0 C0 MEF were infected with MHV before being processed for E Statistical analysis of the samples shown in D performed as described in C F HeLa CEACAM1a cells were infected with MHV nsp2GFP for 7 hr before fractionating collected from the top to the bottom of the gradient and probed with antibodies against forming this experiment three times G HeLa CEACAM1a cells were transiently transfected with a plasmid expressing Cells were fixed at 7 hr p i and processed for IF DMVs and LC3 HA were detected See also Figure S1 Cell Host C 2007 Verheije et al 2008 By IF analysis of MHV infected wild type and Atg7 C0 C0 knockout cells we systematically assessed the presence in DMVs of several conventional protein markers of the early compartment of the secretory pathway essentially confirming that none of them were in the virus induced DMVs Figure S3A and data not shown Only two anti bodies stained the virus induced dsRNA or nsp2 nsp3 positive compartments namely those recognizing EDEM1 and OS 9 E Endogenous LC3 Atg7 Atg7 0 0 20 40 60 80 100 10 20 30 40 50 60 70 GFP LC3 LC3 puncta co localizing with DMVs nsp2 nsp3 merged inset LC3 puncta co localizing with DMVs DMVs GFP LC3 were infected with MHV Srec and processed for IF at 7 hr p i ge of LC3 or GFP LC3 puncta colocalizing with the nsp2 nsp3 signals Error in three independent experiments The asterisk indicates that the two samples IF at 7 hr p i a cell extract on a continuous Optiprep gradient Ten fractions were EDEM1 GFP and LC3 The fractionation profile was confirmed by per C terminally HA tagged nonlipidable LC3 before being infected with MHV with antibodies against nps2 nsp3 and HA respectively Cal et al 2008a Because MHV infection induces a host translational shutoff Raaben et al 2007 the persistence of EDEM1 and OS 9 for several hours confirms the defective clearance of this protein in infected cells Figures 3H S3B and S3C Entrapping of a fraction of cellular OS 9 in the DMVs was unexpected but significant Like EDEM1 OS 9 is a regulator of protein disposal from the ER and it is expressed in two splice variants OS 9 1 and OS 9 2 Bernasconi et al 2008 Christianson et al 2008 Immunode tection of OS 9 in the MHV induced DMVs Figures 3F and 3G led us to verify by subcellular fractionation whether OS 9 local izes to the same LC3 I positive vesicular structures containing EDEM1 in noninfected cells Cal et al 2008a Our analysis showed that at steady state about 20 of OS 9 1 and a smaller amount of OS 9 2 were indeed found in LC3 I positive EDEMo somes which sedimented in fractions 7 9 of a continuous Opti prep gradient whereas other conventional ER chaperones were fully excluded from these fractions Figure S3D Cal et al 2008a Despite their presence in DMVs EDEM1 and OS 9 are not required for MHV replication In fact MHV infectivity was not affected by their knockdown as measured by examining the synthesis of the viral structural N protein the DMV formation by IF and by assessing virus replication by either determining the TCID 50 value of a virus stock on these cells or assaying the luciferase expression levels at different p i time points Figure S4 Taken together these results support the hypothesis that CoV hijack the ERAD tuning machinery to generate the repli cative DMVs LC3 I Is Required for CoV Replication The association of LC3 I with MHV induced DMVs is supported by data showing that these vesicles are decorated with anti LC3 antibodies in cells lacking Atg7 and LC3 II Figure 2D they co sediment with LC3 I but not LC3 II in density gradients Figure 3 Components of the ERAD Tuning Pathway Are Associated A Cell extracts from Atg7 lane 1 and Atg7 C0 C0 lane 2 were separated by SDS PAGE p62 LC3 and tubulin Repetition of the analysis showed no significant differences indicate the relative EDEM1 levels in the knockout cells compared to wild type B Atg7 and Atg7 C0 C0 MEF were metabolically labeled and chased for the times EDEM1 present in each lane was quantified and indicated below each band Repetition Atg7 versus Atg7 C0 C0 MEF C Atg7 MEF were untreated or treated with 100 mM CQ or 1 mM rapamycin rap ages right indicate the relative EDEM1 levels in drug treated cells compared to D Same as B to confirm in a pulse chase radiolabeling experiment that CQ and E and F HeLa cells were infected with MHV Srec and processed for IF at 7 hr p i G Atg7 and Atg7 C0 C0 MEF infected with MHV Srec were fixed at 7 hr p i and H Cell extracts were analyzed by western blot using antibodies against EDEM1 or infected cells compared to control cells and represent the average of two experime See also Figures S2 S3 and S4 Cell Host tial decrease in luciferase levels measured in both single time point and time course experiments Figures 4B and 4C The inhibition of MHV replication observed in LC3 knockdown cells was caused by a defect in DMV biogenesis as no nsp2 and nsp3 signal was detected by IF Figure 4D The initial DMVs and RTCs generated upon MHV infection are necessary for the massive synthesis of extra nsp proteins which in turn leads to the formation of a multitude of additional DMVs and RTCs Consequently a defect in DMV biogenesis results in a severe block of nsp production Crucially back transfection of LC3 knockdown cells with the plasmid expressing C terminally HA tagged nonlipidable LC3 restored MHV replication measured by monitoring N protein synthesis Figure 4E During evolution pathogens have developed molecular devices to exploit conserved cellular pathways to optimally infect replicate and or leave host cells Until now no host protein has been directly implicated in CoV replication The fact that both CoV induced DMV formation and EDEM1 OS 9 turnover rely on analog mechanisms leads us to postulate that CoV hijack the ERAD tuning machinery for the generation of DMVs which provide the membranous support for viral RTCs Based on our data our current working model is that one or more CoV nsps do associate with a still elusive EDEMosome cargo receptor that normally mediates segregation and vesicular export from the ER of EDEM1 and OS 9 Accordingly to what is known about other vesicular transport pathways Sato and Nakano 2007 we postulate that the EDEMosome cargo receptor interacts with subunits of a cytosolic vesicle protein coat nonlipidated LC3 I seems a good candidate It is unlikely that the viral nsps recruit LC3 I at the cytosolic surface of DMVs because LC3 I is associated to EDEMosomes in unin fected cells Cal et al 2008a and because we have not found direct interaction between nsp and LC3 I unpublished data Astonishingly whereas EDEMosomes are transient structures that end their journey in late endosomes and or lysosomes MHV induced DMVs are persistent cytoplasmic organelles Ulasli et al in press Therefore the presence of either nsps or DMVs and western blot membranes probed with antibodies against EDEM1 in the EDEM1 level in Atg7 versus Atg7 C0 C0 MEF The percentages right and represent the average of two experiments indicated before lysis and EDEM1 immuno isolation The residual radiolabeled of the analysis showed no significant differences in t