【病毒外文文献】2007 Mouse hepatitis coronavirus replication induces host translational shutoff and mRNA decay, with concomitant formati

Mouse hepatitis coronavirus replication induces host translational shutoff and mRNA decay with concomitant formation of stress granules and processing bodies Matthijs Raaben 1 Marian J A Groot Koerkamp 2 Peter J M Rottier 1 and Cornelis A M de Haan 1 1 Virology Division Department of Infectious Diseases and Immunology Faculty of Veterinary Medicine Utrecht University Utrecht the Netherlands 2 Microarray Facility UMC Utrecht Department of Physiological Chemistry Utrecht the Netherlands Summary Many viruses including coronaviruses induce host translational shutoff while maintaining synthesis of their own gene products In this study we performed genome wide microarray analyses of the expression patterns of mouse hepatitis coronavirus MHV infected cells At the time of MHV induced host translational shutoff downregulation of numerous mRNAs many of which encode protein translation related factors was observed This downregulation which is reminiscent of a cellular stress response was dependent on viral replication and caused by mRNA decay Concomitantly phosphorylation of the eukaryotic translation initiation factor 2a was increased in MHV infected cells In addition stress granules and processing bodies appeared which are sites for mRNA stalling and degradation respectively We propose that MHV replication induces host trans lational shutoff by triggering an integrated stress response However MHV replication per se does not appear to benefit from the inhibition of host protein synthesis at least in vitro since viral replication was not negatively affected but rather enhanced in cells with impaired translational shutoff Introduction Virus infections often give rise to a shutoff of host cell translation while synthesis of viral proteins is maintained This cellular response provides two advantages for viral replication it facilitates rapid viral protein synthesis and inhibits the production of antiviral proteins Lyles 2000 On the other hand viruses and their hosts share the same translational machinery Therefore global inhibition of protein synthesis can also be regarded as part of an antiviral response of the host aimed to minimize viral replication Coronaviruses CoVs are enveloped positive stranded RNA viruses and are common pathogens in man and animals Their relevance has increased considerably with the recent emergence of new human CoVs HCoVs such as the severe acute respiratory syndrome SARS CoV Drosten et al 2003 HCoV NL63 van der Hoek et al 2004 and HCoV HKU1 Woo et al 2005 CoVs replicate entirely within the cytoplasm of their host cells where they produce a nested set of sub genomic mRNAs containing identical 5 leader sequences and coterminal 3 ends Pasternak et al 2006 These mRNAs which are transcribed via a discon tinuous transcription mechanism are 5 capped and 3 polyadenylated making them structurally equivalent to host cellular mRNAs Sawicki and Sawicki 2005 There fore CoVs must hijack the host translational machinery to produce their own proteins The mouse hepatitis CoV MHV a close relative of the SARS CoV Snijder et al 2003 and the feline infectious peritonitis virus have been shown to induce host transla tional shutoff in susceptible cells Rottier et al 1981 Siddell et al 1981 Hilton et al 1986 M Raaben and C A M de Haan unpubl results The mechanism by which this occurs and how CoVs themselves escape from it is not known As the leader sequence present on all viral mRNAs enhances translation in MHV infected cells it has been suggested that the apparent downregulation of host protein synthesis is not primarily due to inhibition of translation Tahara et al 1994 On the other hand the reduced host mRNA translation in MHV infected cells has also been associated with a decrease in the number and size of polysomes and an increase of inactive single 80S ribosomes Hilton et al 1986 and with cleavage of 28S ribosomal RNA Banerjee et al 2000 Furthermore it has been suggested that a decline in steady state levels found for some host cell mRNAs contributes to the down regulation of host protein translation Hilton et al 1986 Received 22 January 2007 revised 7 March 2007 accepted 27 March 2007 For correspondence E mail c a m dehaan vet uu nl Tel 31 30 2534195 Fax 31 30 2536723 Cellular Microbiology 2007 9 9 2218 2229 doi 10 1111 j 1462 5822 2007 00951 x First published online 8 May 2007 2007 The Authors Journal compilation 2007 Blackwell Publishing Ltd Kyuwa et al 1994 Tahara et al 1994 More recently the expression of the SARS CoV non structural protein 1 nsp1 was reported to induce mRNA degradation and inhibition of host protein synthesis Kamitani et al 2006 Host shutoff is not restricted to virus infected cells but is induced by various stress stimuli Brostrom and Bros trom 1998 For example cells deprived of nutrients inhibit protein synthesis to conserve amino acids for essential metabolic processes In addition cells also shut down protein synthesis in response to endoplasmic reticu lum ER stress heat and oxidative stress These different stress stimuli induce translational shutoff via the increased phosphorylation of eukaryotic initiation factor eIF 2a de Haro et al 1996 In mammalian cells there are at least four eIF2a kinases including GCN2 PKR PERK and HRI which are activated by amino acid star vation double stranded RNA ER stress and haem deple tion respectively Berlanga et al 1999 Harding et al 1999 Kaufman 2000 Lu et al 2001 Upon phosphory lation of eIF2a cytoplasmic structures containing stalled translational preinitiation complexes frequently referred to as stress granules SGs are formed The actual for mation of SGs occurs upon aggregation of the RNA binding proteins TIA 1 T cell internal antigen 1 and TIAR TIA 1 related protein Kedersha et al 1999 SGs are thought to recruit specific mRNA transcripts such as housekeeping protein encoding mRNAs to regulate their translation in adaptation to altered conditions Anderson and Kedersha 2002 SGs are also referred to as triage centres because they sort mRNAs for either storage reinitiation of translation or degradation Anderson and Kedersha 2006 Degradation of mRNA is another way to control gene expression at a post transcriptional level While 3 5 mRNA decay occurs by a complex of exonucleases termed the exosome 5 3 mRNA decay takes place in cytoplasmic foci related to SGs called processing bodies P bodies Anderson and Kedersha 2006 P bodies contain decapping enzymes the exonuclease Xrn1 and components of the RNA induced silencing complex including GW182 which is an RNA binding protein required for microRNA dependent silencing Ingelfinger et al 2002 Eystathioy et al 2003 Cougot et al 2004 Liu et al 2005 The formation of P bodies is also induced in response to different stress stimuli including glucose deprivation and osmotic stress However unlike SGs their assembly does not require phosphorylation of eIF2a Kedersha et al 2005 Teixeira et al 2005 To get more insight into the interference of CoVs with gene expression of their host we investigated the global mRNA profiles in MHV infected cells by using genome wide microarray analysis At the time of host shutoff sig nificant downregulation of many mRNAs particularly of those encoding translation related factors was observed This downregulation which is reminiscent of a cellular stress response was shown to be dependent on viral replication and to result from mRNA decay At the same time increased phosphorylation of eIF2a and the assem bly of SGs and P bodies were observed suggesting that these cellular structures play an important role in the host translational shutoff and mRNA decay respectively MHV replication was not negatively affected but rather enhanced in mouse embryo fibroblast MEF cells in which host translational shutoff and SG formation were impaired by mutation Apparently virus replication does not benefit at least in vitro from the induced translational shutoff Results Kinetics of MHV induced host translational shutoff Before starting with the microarray analyses the kinetics of the MHV induced host translational shutoff was deter mined for the experimental conditions used To this end metabolic labelling of MHV infected LR7 cells was per formed at different time points post infection The results shown in Fig 1A clearly demonstrated that host trans lation was shut down at 6 but not at 4 h post infection while the viral spike S nucleocapsid N and membrane M proteins were clearly visible at 6 h and at later time points post infection Thus between 4 and 6 h post infec tion a shift occurred from host to viral protein synthesis This conclusion was corroborated by quantification of the radioactivity in the gel Fig 1B The data show that there is a continuous rise in expression of the viral protein S N and M with the largest increase appearing between 4 and 6 h post infection The amount of radioactivity in the regions between the viral proteins representative of host protein synthesis were also quantified and confirmed the induction of host shutoff between 4 and 6 h post infection Next the levels of genomic viral RNA in the MHV infected cells were determined Total RNA was isolated at different time points post infection and viral RNA was quantified using quantitative reverse transcription poly merase chain reaction RT PCR The results show that viral RNA accumulated during infection reaching a steady state level at approximately 8 h post infection Fig 1C Consistent with the results of the metabolic labelling experiment the largest relative increase in viral RNA levels was observed between 4 and 6 h post infection Fig 1D Altogether the results demonstrate that under the experimental conditions used host trans lational shutoff is induced in MHV infected LR7 cells between 4 and 6 h post infection and is accompanied by a relatively large increase in viral RNA and protein synthesis Stress induced mRNA decay by MHV 2219 2007 The Authors Journal compilation 2007 Blackwell Publishing Ltd Cellular Microbiology 9 2218 2229 Microarray analysis of MHV infected LR7 cells reveals large scale mRNA degradation To get more insight into the MHV induced host transla tional shutoff we investigated the global mRNA profiles in infected cells by using genome wide microarray analysis To this end parallel cultures of LR7 cells were either mock infected or infected with MHV A59 and total RNA was isolated at 4 and 6 h post infection As will be pub lished elsewhere at 6 h post infection viral mRNA consti tutes approximately 40 of the total mRNA content in infected cells By removing these unwanted transcripts using the oligo capture technique we reduced non specific hybridization by approximately 80 The mRNAs in the infected and uninfected samples were amplified labelled with either Cy3 or Cy5 and hybridized to the arrays after which they were scanned and analysed At 4 h post infection relatively few changes in the expression profile were observed Fig 2A However at 6 h post infec tion a significant number of genes appeared to be altered in their expression Fig 2B Besides the apparent upregu lation of several mRNAs a large number of mRNAs 678 was significantly downregulated upon infection This set of mRNAs is represented by the yellow dots in the different panels of Fig 2 Actinomycin D ActD an inhibitor of cellular transcription but not of CoV replication Robb and Bond 1979 Lai et al 1981 was used to establish whether the observed mRNA downregulation was the Fig 1 Kinetics of the MHV induced host translational shutoff A LR7 cells were infected with MHV moi 10 and metabolically labelled for 15 min starting at the indicating time points Cell lysates were processed and subjected to SDS PAGE as described in Experimental procedures Positions of the viral proteins S N and M are indicated B The amount of radioactivity in the gel was quantified with a PhosphorImager For each time point the amount of radioactivity in the MHV structural proteins S N and M was combined viral expression For the host proteins the amount of radioactivity in the regions between the MHV proteins was quantified host expression The data are presented as relative expression 2 h post infection 1 C and D Genomic viral RNA levels in MHV infected LR7 cells moi 10 were measured by quantitative RT PCR at the indicated time points post infection The data are presented as relative viral RNA levels 12 h post infection 1 in C or as the fold change increase relative to the previous time point in D Error bars indicate the standard deviations n 3 2220 M Raaben M J A Groot Koerkamp P J M Rottier and C A M de Haan 2007 The Authors Journal compilation 2007 Blackwell Publishing Ltd Cellular Microbiology 9 2218 2229 result of a transcriptional or a post transcriptional mecha nism Treatment of both infected and uninfected cells with ActD did not affect the downregulation of mRNAs indicat ing that the observed decrease is the result of post transcriptional mRNA degradation Fig 2C To study the specificity of this virus induced mRNA degradation the effect of ActD treatment was analysed in the absence of a viral infection Treatment of cells with ActD or with other transcriptional inhibitors induces the downregulation of short lived mRNAs Cheadle et al 2005 Indeed down regulation of many mRNAs was observed in ActD treated cells when compared with mock treated cells Fig 2D However the mRNA population downregulated by ActD treatment clearly differed from the mRNAs downregulated in response to MHV infection at 6 h post infection the latter ones are represented by the yellow dots In order to determine the role of virus replication in the mRNA decay cells were inoculated with fusion competent replication incompetent UV inactivated virus The results show that the MHV induced downregulation of mRNA shown again as yellow dots is replication dependent as it was not observed after inoculation with the UV inactivated virus Fig 2E By using Gene Ontology GO databases 240 of the Fig 2 Replication dependent mRNA decay in MHV infected cells as determined by microarray analyses LR7 cells were infected with MHV moi 10 Total RNA was isolated from MHV infected and mock infected cells and processed for microarray analysis as described in Experimental procedures The scatter plots display the average expression values from independent dye swap hybridizations for each gene present on the arrays Red spots represent upregulated gene transcripts while green spots represent downregulated transcripts The dashed lines indicate the twofold change cut off Transcripts downregulated at 6 h post infection in B are represented by the yellow dots throughout the figure Note that these transcripts are significantly changed at 6 h post infection according to SAM applying a false discovery rate of 1 and a cut off at a twofold change described in Experimental procedures A The expression profile of MHV infected cells compared to mock infected cells at 4 h post infection average of four arrays two independent dye swaps n 4 B The expression profile of MHV infected cells compared with mock infected cells at 6 h post infection n 6 C The expression profile of MHV infected cells compared with mock infected cells at 6 h post infection n 4 Both MHV and mock infected cells were treated with ActD for 7 h from 1 till 6 h post infection D The expression profile of ActD treated cells compared with mock treated cells Non infected cells were treated or mock treated with ActD for 7 h n 1 E The expression profile of cells treated with UV inactivated viral particles compared with mock treated cells at 6 h post inoculation n 1 Stress induced mRNA decay by MHV 2221 2007 The Authors Journal compilation 2007 Blackwell Publishing Ltd Cellular Microbiology 9 2218 2229 678 mRNAs downregulated at 6 h post infection could be annotated by their GO terms for biological functions processes see Supplementary Table S1 Strikingly 50 of the mRNAs that could be annotated encode protein translation related factors ribosome biogenesis factors translation initiation elongation associated factors In summary the results show that at the time of virus induced host translational shutoff significant downregula tion of many mRNAs encoding largely translation related factors was observed This downregulation was found to be dependent on viral replication and to result from mRNA decay Transcriptional upregulation of mRNAs Comparison of the transcriptional profiles of MHV infected cells in the absence or presence of ActD Fig 2B and C revealed that the mRNAs from relatively few genes were upregulated at the transcriptional level as the majority of the upregulated mRNAs were obtained for both conditions As approximately 20 of the viral RNA present in the LR7 cells at 6 h post infection was not removed by the oligo capture technique M Raaben and C A M de Haan unpubl results a large number of the mRNAs upregulated in the presence of ActD are likely to result from cross hybridization of the remaining viral RNA Indeed this was confirmed for some hits using quantitative RT PCR analy sis data not shown However we cannot exclude that some of these hits might also result from increased mRNA stability upon virus infection a subject of further research In order to exclude potential false positive hits the genel ists of the experiments performed in the absence or pres ence of ActD were compared As a result 243 genes were excluded from the analysis since they were upregulated under both experimental conditions leaving 40 genes that were specifically upregulated at the transcriptional level in MHV infected LR7 cells at 6 h post infection The genes identified were clustered according to their GO terms for biological processes see Supplementary Table S2 Out of the 40 genes we could annotate 29 genes Examples of genes of which the expression was induced were those encoding inflammatory cytokines Cxcl1 Cxcl2 a marker of the unfolded protein response Herpud1 and dual specificity phosphatase 1 Dusp1 In addition several genes involved in regulation of transcription were differen tially expressed The expression of several genes at 4 and 6 h post infection was confirmed by quantitative RT PCR see Supplementary Table S3 Phosporylation of eIF2a and the formation of stress granules and processing bodies The observed downregulation of numerous mRNAs many of which encode protein translation related factors is reminiscent of a cellular stress response Recently SGs have been associated with the initiation of host transla tional shutoff during stress A key aspect in the formation of these cytoplasmic structures which contain stalled translation preinitiation complexes is the phosphorylation of the translation initiation factor eIF2a at serine 51 which results in the aggregation of the mRNA binding proteins TIA 1 and TIAR both markers for SGs Kedersha et al 1999 Kedersha et al 2005 Phosphorylation of eIF2a was analysed in MHV infected LR7 cells at 4 and 6 h post infection i e before and at the time of host translational shutoff respectively Fig 3A Mock infected cells and cells treated for 30 min with 0 5 mM sodium arsenite were taken along as controls Sodium arsenite has been shown to induce phosphorylation of eIF2a at serine 51 McEwen et al 2005 The expression levels and phos phorylation status of eIF2a in all samples were visualized by immunoblotting using antibodies against total eIF2a and phosphorylated eIF2a eIF2aP respectively Quanti tative analysis revealed that the levels of phosphorylated eIF2a corrected for the total amount of eIF2a inMHV infected cells were reproducibly 2 5 fold higher at 6 h post infection compared with mock infected cells while at 4 h post infection no increase could be observed Subsequently we investigated whether SGs were formed in LR7 cells upon MHV infection As a positive control LR7 cells were incubated with sodium arsenite as described above The cells were fixed at the indicated time points post infection and processed for immunofluo rescence using anti TIAR antibodies Indeed cytoplasmic foci contai