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SARS Coronavirus Replication Transcription Complexes Are Membrane Protected and Need a Host Factor for Activity In Vitro Martijn J van Hemert 1 Sjoerd H E van den Worm 1 Ke vin Knoops 1 2 A Mieke Mommaas 2 Alexander E Gorbalenya 1 Eric J Snijder 1 1Molecular Virology Laboratory Department of Medical Microbiology Leiden University Medical Center Leiden The Netherlands 2Section Electron Microscopy Department of Molecular Cell Biology Leiden University Medical Center Leiden The Netherlands Abstract SARS coronavirus SARS CoV replication and transcription are mediated by a replication transcription complex RTC of which virus encoded non structural proteins nsps are the primary constituents The 16 SARS CoV nsps are produced by autoprocessing of two large precursor polyproteins The RTC is believed to be associated with characteristic virus induced double membrane structures in the cytoplasm of SARS CoV infected cells To investigate the link between these structures and viral RNA synthesis and to dissect RTC organization and function we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system Mainly positive strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro All RTC activity enzymatic and putative membrane spanning nsps and viral RNA cosedimented with heavy membrane structures Furthermore the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity Newly synthesized subgenomic RNA appeared to be released while genomic RNA remained predominantly associated with the RTC containing fraction RTC activity was destroyed by detergent treatment suggesting an important role for membranes The RTC appeared to be protected by membranes as newly synthesized viral RNA and several replicase transcriptase subunits were protease and nuclease resistant and became susceptible to degradation only upon addition of a non ionic detergent Our data establish a vital functional dependence of SARS CoV RNA synthesis on virus induced membrane structures Citation van Hemert MJ van den Worm SHE Knoops K Mommaas AM Gorbalenya AE et al 2008 SARS Coronavirus Replication Transcription Complexes Are Membrane Protected and Need a Host Factor for Activity In Vitro PLoS Pathog 4 5 e1000054 doi 10 1371 journal ppat 1000054 Editor Ralph S Baric University of North Carolina United States of America Received January 16 2008 Accepted April 1 2008 Published May 2 2008 Copyright C223 2008 van Hemert 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 Funding This work was supported in part by grants from the Council for Chemical Sciences of the Netherlands Organization for Scientific Research NWO CW grants 700 52 306 and 700 55 002 and by the European Commission in the context of the activities of the Euro Asian SARS DTV Network SP22 CT 2004 511064 Funding sources had no role in study design data collection data analysis or writing the paper Competing Interests The authors have declared that no competing interests exist E mail e j snijder lumc nl These authors contributed equally to this work Introduction Following infection and genome translation positive strand RNA RNA viruses establish a cytoplasmic enzyme complex that directs the amplification and expression of their genome The viral RNA dependent RNA polymerase RdRp is the central enzyme of this replication transcription complex RTC but it also may include other viral non structural proteins nsps and host factors that cooperate to synthesize viral RNA Over the past decade it has become clear that RNA virus RTCs are invariably associated with virus induced membrane structures which are poorly characterized but presumably provide a framework for RNA synthesis by facilitating the concentration and cooperation of viral macromolecules on a dedicated membrane surface They may also protect the viral RNA from nucleases in the cytoplasm of the host cell aid in shielding the double stranded RNA intermediates of virus replication from the host cell s innate immune system or contribute to the coordination of the viral life cycle in time and space These membrane bound RTCs are the molecular machines that drive the RNA synthesis and evolution of RNA viruses Clearly unraveling their structure and function will be critical to understand the biochemistry of RNA virus replication and develop novel antiviral control strategies The RTC of coronaviruses including that of SARS coronavirus SARS CoV the causative agent of the life threatening severe acute respiratory syndrome for a review see reference 1 stands out for a number of reasons First at 27 32 kb the polycistronic coronavirus genome is by far the largest genome among currently known RNA viruses 2 Second the viral RNA synthesizing machinery not only amplifies the genome but also directs the synthesis of a set of subgenomic sg mRNAs eight in the case of SARS CoV RNA2 to RNA9 to express the viral accessory and structural protein genes The latter are produced from a corresponding set of subgenome length negative strand RNAs which derive from discontinuous negative strand RNA synthesis 3 4 Third the viral replicase transcriptase which will be referred to as replicase for brevity is of unprecedented size and complexity 5 6 It is produced by translation of the partly PLoS Pathogens www plospathogens org 1 May 2008 Volume 4 Issue 5 e1000054 overlapping open reading frames ORF 1a and 1ab with expression of the latter requiring a 1 ribosomal frameshift near the end of ORF1a In this manner SARS CoV genome translation yields the large replicase polyproteins pp1a 4 382 aa and pp1ab 7 073 aa Extensive autoproteolytic processing mediated by two ORF1a encoded protease domains 7 10 ultimately generates 16 nsps 5 6 11 12 These include key replicative enzymes e g the nsp12 RdRp 13 and the nsp13 helicase 14 a variety of subunits containing presumed accessory functions for viral RNA synthesis e g the nsp8 primase 15 16 nsp14 exoribonuclease 17 18 and nsp15 endoribonuclease Nen doU 19 22 and several predicted multi spanning membrane proteins nsp3 nsp4 and nsp6 23 24 that presumably modify cellular endomembranes and target the RTC to this scaffold Immunofluorescence microscopy previously revealed that newly synthesized SARS CoV RNA and several nsps colocalize in perinuclear foci in SARS CoV infected cells 8 14 25 27 Elec tron microscopy established the presence of typical paired membranes membrane whorls and double membrane vesicles DMVs which labeled for nsps 26 29 and viral RNA 27 and were therefore proposed to carry the SARS CoV RTC The endoplasmic reticulum ER was identified as the most likely membrane donor 26 and recent electron tomography studies indeed revealed a network of SARS CoV induced membrane structures that is continuous with this organelle Knoops et al in preparation In the past four years substantial progress has been made in the characterization of individual replicase subunits using enzymatic assays reverse and classical genetics bioinformatics and structural studies However the composition and mechanistics of the native ribonucleoprotein complexes in which these different components interact to drive coronavirus replication and tran scription have remained completely uncharacterized thus far We therefore set out to isolate active RTCs from SARS CoV infected cells and used those to develop an in vitro system that faithfully reproduced the synthesis of both genomic and sg RNAs mainly of positive polarity RTC activity cosedimented with newly synthe sized viral RNA and several replicase subunits in a dense membrane fraction containing structures that could be labeled for nsp3 and nsp4 The in vitro activity of the pelleted RTC depended on the presence of a cytoplasmic host factor Furthermore RTC activity was destroyed by addition of non ionic detergents which also released replicase subunits and mainly sg RNA from the membrane fraction Protease and nuclease protection experiments indicated that viral RNA and nsps were protected by membranes thus further substantiating the functional connection between SARS CoV RNA synthesis and virus induced membrane structures that appear to be essential for RTC activity Results Isolation of active SARS CoV RTCs In order to characterize isolated SARS CoV RTCs we developed an in vitro RNA synthesis assay IVRA to study their activity in vitro In this reaction the incorporation of a 32 P CTP into viral RNA was analyzed in a mixture containing NTPs Mg 2 an ATP regenerating system and an inhibitor of cellular transcription Actinomycin D The RTC activity in cytoplasmic extracts prepared from SARS CoV infected Vero E6 cells pro duced a set of 32 P labeled RNA molecules with sizes correspond ing to those of the SARS CoV genome and all eight sg RNAs Fig 1 These products were not detected when using mock infected cell lysates Fig 1A mock demonstrating that SARS CoV RTC activity was responsible for their synthesis Reaction conditions were optimized by varying several parameters including the composition of the reaction mixture incubation time temperature and the Mg 2 concentration Fig 1 and data not shown In a time course experiment in vitro synthesized viral RNA accumulated up to 100 min into the reaction Fig 1A after which a decrease was observed probably due to declining RTC activity in combination with continued RNA degradation by cellular nucleases The optimal reaction temperature was 30uC Fig 1B RTC activity was strongly dependent on the Mg 2 concentration and was maximal when 2 mM of Mg 2 was added to the reaction Fig 1C Manganese could not replace Mg 2 as IVRAs containing Mn 2 only yielded a ladder of small radiolabeled RNA molecules with aberrant sizes Fig 1D suggesting an effect on RdRp processivity Addition of ionic SDS and deoxycholate DOC or non ionic detergents Nonidet P40 NP 40 and Triton X 100 TX 100 to the post nuclear supernatant PNS prior to the IVRA completely abolished the accumulation of radiolabeled viral RNA suggesting that the integrity of membranes is an important factor for SARS CoV RTC activity Fig 1D To determine the polarity of the in vitro produced RNAs the 32 P labeled products of an IVRA were hybridized to a membrane containing immobilized RNA probes specific for SARS CoV positive or negative stranded RNA Fig 1E A strong hybridiza tion with the positive strand specific probe was observed demonstrating that the RTC mainly synthesized RNA of positive polarity in vitro After longer exposure times a small quantity of radiolabeled material hybridizing to the negative strand specific probe became visible but a similar signal was observed with the negative control RNA Fig 1E This indicated that the quantity of in vitro synthesized negative stranded RNA was very small less than 2 of the total RNA which is in line with the large excess of positive over negative strands that is commonly observed in vivo Protein synthesis is not required for RTC activity in vitro To assess whether protein synthesis occurred during IVRAs we determined whether 35 S labeled amino acids were incorporated Author Summary The SARS coronavirus SARS CoV which causes the life threatening severe acute respiratory syndrome replicates in the cytoplasm of infected host cells A critical early step in the SARS CoV life cycle is the formation of a replication transcription complex RTC that drives viral genome replication and subgenomic mRNA synthesis Virus encod ed enzymes form the core of this RTC which is believed to be associated with characteristic virus induced membrane structures derived from modified host cell membranes To investigate the connection between these membrane structures and SARS CoV RNA synthesis and to character ize RTC composition and function we isolated these complexes and developed the first in vitro assay to study their activity SARS CoV genomic RNA and all eight subgenomic mRNAs were synthesized in this in vitro reaction By centrifugation RTC activity could be isolated from the cytoplasm together with membrane structures viral enzymes and RNA The activity of these isolated RTCs was dependent on a cytoplasmic host factor RTC activity was destroyed by detergent treatment suggesting a critical role for membranes that appeared to protect the complex against protease and nuclease digestion Our data establish a functional connection between viral RNA synthesis and intracellular membranes and show that host factors play a crucial role in SARS CoV RNA synthesis SARS Coronavirus RNA Synthesis and Membranes PLoS Pathogens www plospathogens org 2 May 2008 Volume 4 Issue 5 e1000054 into proteins during a 100 min reaction When using the PNS of uninfected cells SDS PAGE revealed a smear of 35 S labeled material Fig 2A lane 2 These products were absent when the PNS was heated to 96uC for 5 min prior to the assay Fig 2A lane 1 suggesting they resulted from translation under IVRA conditions When using the PNS of SARS CoV infected cells we observed incorporation of radiolabel also into a set of discrete polypeptides Fig 2A lane 4 including species with sizes matching those of the SARS CoV membrane M and nucleocap sid N proteins This was likely due to the fact that the lysate contained large amounts of the sg mRNAs encoding these proteins possibly in combination with the virus induced shut off of host cell translation 30 Protein synthesis was completely inhibited when the translation inhibitors cycloheximide or puromycin were present during the IVRA Fig 2A lanes 5 and 6 but this did not affect in vitro RTC activity since the quantity of radiolabeled RNA products was unchanged Fig 2B The activity of isolated RTCs depends on a cytoplasmic host factor To further characterize the active RTC the PNS of SARS CoV infected cells was subjected to differential centrifugation A Figure 1 In vitro RNA synthesizing activity of SARS CoV RTCs Incorporation of a 32 P CTP into viral RNA in IVRAs with PNS from SARS CoV or mock infected cells was analyzed by denaturing formaldehyde agarose gel electrophoresis followed by PhosphorImager analysis To optimize reaction conditions the reaction time A temperature B and Mg 2 concentration C were varied as indicated above the lanes Except for the parameter that was varied standardized conditions were used as described in Materials and Methods The mock lane contains the reaction products from a standard 100 min IVRA performed with a PNS from mock infected cells D IVRAs with 2 mM Mn 2 instead of Mg 2 or with 0 1 SDS 0 03 DOC 0 01 NP 40 or 0 01 TX 100 added as indicated above the lanes The position of RNAs 1 9 are indicated and were determined by including a lane hyb with RNA isolated from SARS CoV infected cells that was hybridized with a probe complementary to the 39 end of all viral RNAs The seemingly different ratios between genomic and sgRNA when IVRA results are compared with the hybridization data are explained by the fact that the former reflect the size dependent incorporation of label whereas the latter show molar abundance The molar ratios of IVRA products calculated using the C content of each RNA species are similar to those in infected cells E Analysis of the polarity of in vitro synthesized SARS CoV RNA A membrane containing immobilized SARS CoV RNA probes of positive 39 TR or negative polarity 39 TR was hybridized with the 32 P labeled RNA products of an IVRA RNA probes derived from the equine arteritis virus genome ctrl a or its complement ctrl b were included as negative controls See Materials and Methods for probe details A short and a long exposure of the same hybridization are shown doi 10 1371 journal ppat 1000054 g001 SARS Coronavirus RNA Synthesis and Membranes PLoS Pathogens www plospathogens org 3 May 2008 Volume 4 Issue 5 e1000054 10 0006g supernatant fraction S10 showed no RTC activity Fig 3 lane 2 but only a trace amount of the original activity was recovered in the 10 0006g pellet fraction P10 Fig 3 lane 4 Surprisingly RTC activity in this P10 fraction could be largely restored by adding an aliquot of the cytoplasmic S10 fraction Fig 3 lane 5 An S10 fraction prepared from mock infected cells was equally capable of restoring the RTC activity in P10 indicating that a cytoplasmic host factor was required Fig 3 lane 6 Routinely about 50 of the RTC activity that was originally present in the PNS could be recovered in the P10 fraction in assays supplemented with S10 Remarkably virtually all replicative activity was lost while transcription was only 2 to 3 fold decreased in the P10 fraction depleted of the host factor Fig 3 lane 4 The sedimentation of the RTC activity at 10 0006g suggests that it is associated with heavy membrane structures RTC activity cosediments with membrane structures The P10 fraction which contained the bulk of RTC activity was analyzed by electron microscopy negative staining in combination with an immunogold labeling for the putative transmembrane proteins nsp3 nsp4 and nsp6 Fig 4 and data not shown Clusters of vesicles with diameters between 100 and 350 nm were observed which appeared to be associated with more tubular or flattened membrane structures A strong immunolabeling of these structures for SARS CoV nsp3 Fig 4A and nsp4 Fig 4B was observed Membrane structures immuno Figure 2 Protein synthesis is not required for RTC activity in vitro A Incorporation of 35 S labeled amino acids into proteins during a 100 min IVRA Proteins were separated by SDS PAGE and incorporation of radiolabel was visualized by phosphorimaging of the dried gel PNS from either uninfected or SARS CoV infected cells was used which was either untreated 2 or heated to 96uC prior to the reaction as indicated above the lanes Reactions were performed in the absence 2 or presence of the translation inhibitors cycloheximide CHX or puromycin PUR The positions of protein size markers are indicated on the left and the arrows on the right indicate the positions of polypeptides with sizes matching those of SARS CoV membrane M and nucleocapsid N proteins B The effect of translation inhibitors on in vitro RTC activity IVRAs were performed in the absence 2 or presence of cycloheximide CHX or puromycin PUR as indicated above the lanes Reaction products were analyzed as described in the legend to Fig 1 doi 10 1371 journal ppat 1000054 g002 Figure 3 The activity of isolated RTCs is dependent on a cytoplasmic host factor The PNS of SARS CoV infected or uninfect ed cells was subjected to differential centrifugation after which IVRAs were performed using the PNS lane 1 10 0006g pellet P10 and supernatant S10 fractions either on a single fraction or combinations of them as indicated above the lanes Reaction products were analyzed as described in the legend to Fig 1 doi 10 1371 journal ppat 1000054 g003 SARS Coronavirus RNA Synthesis and Membranes PLoS Pathogens www plospathogens org 4 May 2008 Volume 4 Issue 5 e1000054 reactive for nsp3 Fig 4C or nsp4 data not shown were not detected in a control P10 fraction prepared from mock infected cells Occasionally double membranes could be distinguished Fig 4B arrow These observations are consistent with the notion that the P10 fraction is enriched for SARS CoV induced nsp containing membrane structures that have been documented in infected cells Product specific differences in the release of SARS CoV RNAs from the RTC The distribution of newly synthesized SARS CoV RNAs between the RTC containing P10 and cytoplasmic S10 fractions was analyzed by fractionation of PNS after an IVRA Fig 5A The bulk 76 of newly made genomic RNA was recovered from the P10 fraction suggesting it remained associated with the heavy membrane structures In contrast newly synth