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Cell Host Response to Infection with Novel Human Coronavirus EMC Predicts Potential Antivirals and Important Differences with SARS Coronavirus Laurence Josset a Vineet D Menachery b c Lisa E Gralinski b c Sudhakar Agnihothram b c Pavel Sova a Victoria S Carter a Boyd L Yount b c Rachel L Graham b c Ralph S Baric b c Michael G Katze a Department of Microbiology School of Medicine University of Washington Seattle Washington USA a Department of Epidemiology b and Department of Microbiology and Immunology c University of North Carolina at Chapel Hill Chapel Hill North Carolina USA L J and V D M contributed equally to this work ABSTRACT A novel human coronavirus HCoV EMC was recently identified in the Middle East as the causative agent of a severe acute respiratory syndrome SARS resembling the illness caused by SARS coronavirus SARS CoV Although derived from the CoV family the two viruses are genetically distinct and do not use the same receptor Here we investigated whether HCoV EMC and SARS CoV induce similar or distinct host responses after infection of a human lung epithelial cell line HCoV EMC was able to replicate as efficiently as SARS CoV in Calu 3 cells and similarly induced minimal transcriptomic changes before 12 h postin fection Later in infection HCoV EMC induced a massive dysregulation of the host transcriptome to a much greater extent than SARS CoV Both viruses induced a similar activation of pattern recognition receptors and the interleukin 17 IL 17 pathway but HCoV EMC specifically down regulated the expression of several genes within the antigen presentation pathway including both type I and II major histocompatibility complex MHC genes This could have an important impact on the ability of the host to mount an adaptive host response A unique set of 207 genes was dysregulated early and permanently throughout infec tion with HCoV EMC and was used in a computational screen to predict potential antiviral compounds including kinase inhib itors and glucocorticoids Overall HCoV EMC and SARS CoV elicit distinct host gene expression responses which might im pact in vivo pathogenesis and could orient therapeutic strategies against that emergent virus IMPORTANCE Identification of a novel coronavirus causing fatal respiratory infection in humans raises concerns about a possible widespread outbreak of severe respiratory infection similar to the one caused by SARS CoV Using a human lung epithelial cell line and global transcriptomic profiling we identified differences in the host response between HCoV EMC and SARS CoV This enables rapid assessment of viral properties and the ability to anticipate possible differences in human clinical responses to HCoV EMC and SARS CoV We used this information to predict potential effective drugs against HCoV EMC a method that could be more generally used to identify candidate therapeutics in future disease outbreaks These data will help to generate hy potheses and make rapid advancements in characterizing this new virus Received 5 March 2013 Accepted 12 April 2013 Published 30 April 2013 Citation Josset L Menachery VD Gralinski LE Agnihothram S Sova P Carter VS Yount BL Graham RL Baric RS Katze MG 2013 Cell host response to infection with novel human coronavirus EMC predicts potential antivirals and important differences with SARS coronavirus mBio 4 3 e00165 13 doi 10 1128 mBio 00165 13 Invited Editor Michael Buchmeier University of California Irvine Editor Michael Buchmeier University of California Irvine Copyright 2013 Josset et al This is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial ShareAlike 3 0 Unported license which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original author and source are credited Address correspondence to Michael G Katze honey u washington edu I n September 2012 a novel human coronavirus HCoV EMC was reported to health authorities from two cases of acute respi ratory syndrome with renal failure 1 4 The most recent update by the WHO identified a total of 17 confirmed cases of human infection including 11 deaths suggesting a mortality rate of 65 5 All of these individuals had a history of recent travel to the Middle East Identification of clusters of coronavirus cases indi cates that HCoV EMC can be transmitted from human to human 6 and raises concern about a possible outbreak of this virus similar to the one caused by a related virus the severe acute respi ratory syndrome related coronavirus SARS CoV in 2002 2003 SARS CoV originating in China spread throughout Asia and to other continents and affected more than 8 000 people 7 8 The overall mortality during the outbreak was estimated at 9 6 7 While the mortality rate of HCoV EMC cannot be assessed with certainly it could be more pathogenic than SARS CoV HCoV EMC belongs to the genus betacoronavirus as does SARS CoV However HCoV EMC is more closely related to the bat coronaviruses HKU4 and HKU5 lineage 2C than it is to SARS CoV lineage 2B 2 9 Less than 50 amino acid sequence identity is conserved in the replicase domains between SARS CoV and HCoV EMC Another important difference between the two viruses is that they do not use the same host cell receptor for infection 10 Indeed it was clearly shown that human angiotensin converting receptor 2 hACE2 used by SARS CoV is not the HCoV EMC receptor 10 Dipeptidyl peptidase 4 was recently identified as the HCoV EMC receptor 11 This receptor is conserved among different species such as bats and humans RESEARCH ARTICLE May June 2013 Volume 4 Issue 3 e00165 13 mbio asm org 1 mbio asm org on September 13 2015 Published by mbio asm org Downloaded from partially explaining the large host range of HCoV EMC This was somewhat surprising as coronaviruses generally show strict host specificity While recent identification of the crystal structure of HCoV EMC protease suggests that a wide spectrum CoV protease inhib itor could block the catalytic site 12 there is currently no proven antiviral treatment for HCoV EMC Viruses rely on host factors to replicate and often hijack cellular processes initiated in response to infection to ensure efficient replication 13 Targeting cellular responses has been shown to inhibit viral replication 13 14 Furthermore immunomodulatory drugs that reduce the exces sive host inflammatory response to respiratory viruses as seen with influenza virus infections have therapeutic benefit reviewed in reference 15 Several genome based drug repurposing strate gies successfully identified known drugs that could be reused to treat lung cancer inflammatory bowel disease 16 and influenza virus infection 14 Such an approach has the advantage of accel erating treatment availability which could be crucial in case of an outbreak of an emerging pathogen Overall differences in viral sequences host cell receptor and host range indicate that HCoV EMC and SARS CoV may have distinct strategies for interacting with their hosts This fact could impact treatment strategies To begin to assess this question we compared the host response of human cells to HCoV EMC and SARS CoV infection using global transcriptomic profiling Our goal was to gain a rapid and comprehensive assessment of the host response to HCoV EMC infection that could guide research on this emerging virus Importantly we used this information to computationally predict antiviral treatment and identified a broad down regulation of the antigen presentation pathway that may be important in vivo for the development of an adaptive im mune response RESULTS SARS CoV and HCoV EMC have similar replication kinetics but different cytopathic effects in a human epithelial cell line To characterize HCoV EMC we sought a cell line that mimics the human airway Calu 3 cells derived from epithelial cells lining the human conducting airway can be differentiated into polarized ciliated cells and permit robust replication of several respiratory viruses including SARS CoV influenza A virus and respiratory syncytial virus RSV 17 20 Therefore Calu 3 cells were in fected with HCoV EMC using a multiplicity of infection MOI of 5 and results were compared with those for Calu 3 cells infected with SARS CoV Urbani at the same MOI The viruses replicated to similar levels with both peaking atH1102210 7 PFU at 24 h postinfec tion hpi Fig 1 While SARS CoV has been previously shown to maintain steady replication and cell viability to 72 hpi 19 HCoV EMC induced substantial cytopathic effect at 18 to 24 hpi with significant cell rounding and detaching Together these data suggest that although HCoV EMC and SARS CoV exhibit similar replication kinetics they elicit different host responses in lung epithelial cells HCoV EMC induces earlier and different transcriptional changesthanSARS CoV To assess the global host transcriptional response following infection with HCoV EMC samples of in fected Calu 3 cells were collected throughout a 24 h time course postinfection Genes that were differentially expressed DE com pared to time matched mock infected controls were determined using the statistical cutoff of a q value of H110210 01 and an absolute log 2 FC of H110221 These genes were compared to DE genes from Calu 3 cells infected with SARS CoV across a 72 h time course using the same statistical cutoff Fig 2 As previously observed 19 SARS CoV is able to replicate actively with a surprisingly small number of significant transcrip tional changes before 24 hpi from 5 DE genes at 0 hpi to 47 genes at 12 hpi Fig 2A Similarly HCoV EMC induced fewer tran scriptional changes before 12 hpi than after However HCoV EMC replication at early times postinfection induced more changes than SARS CoV with the number of DE genes ranging from 28 at 0 hpi to 206 genes at 12 hpi Fig 2A At later times postinfection a massive host response was observed during HCoV EMC infection with 6 532 DE genes at 18 hpi and 11 664 genes at 24 hpi while SARS CoV induced changes of only 792 genes at 24 hpi with maximum changes at 48 and 54 hpi of 6 496 and 6 498 genes respectively To evaluate the similarity of transcriptional dysregulation be tween HCoV EMC and SARS CoV we determined the percent age of overlap among DE genes changing in the same direction at each time point Fig 2B The intersection between up or down regulated DE genes for each condition was calculated separately and then averaged to determine the percentage of intersecting genes Fig 2B The overlap between signatures at late times postinfection for a single virus was very high for example 89 of the genes DE by HCoV EMC at 18 hpi were also DE at 24 hpi and on average 77 of the DE genes at 24 hpi with SARS CoV were also DE at later times postinfection However the overlap between SARS CoV and HCoV EMC at the same time point was low for example only 3 of the DE genes in response to HCoV EMC at 24 hpi were also DE by SARS CoV at this time point Of note the intersection of DE genes between the two infections was higher when later times postinfection for SARS CoV were compared with earlier time points for HCoV EMC On average 22 of the FIG 1 HCoV EMC replicates at a level similar to that of SARS CoV in human epithelial cells Triplicate wells of Calu 3 2B4 cells were infected with HCoV EMC MOI 5 Medium from each well was collected and analyzed by plaque assay for viral growth kinetics in VeroE6 cells The corresponding cells were harvested for transcriptomic analysis SARS CoV titers after infection of Calu 3 2B4 cells at an MOI of 5 were determined using the same method 19 The error bars represent the standard deviations among triplicate cell samples Josset et al 2 mbio asm org May June 2013 Volume 4 Issue 3 e00165 13 mbio asm org on September 13 2015 Published by mbio asm org Downloaded from HCoV EMC signatures from 12 to 24 hpi overlapped the signa tures of SARS CoV from 36 to 72 hpi Together these results indicate that HCoV EMC induced both a more robust and a largely different host response compared to that induced by SARS CoV at similar times postinfection However changes in duced from 12 to 24 hpi after HCoV EMC were more similar to changes induced late after SARS CoV infection 24 to 72 hpi HCoV EMC massively dysregulates the host transcriptome at late times postinfection At late times postinfection HCoV EMC induced drastic changes in the host transcriptome with 12 392 DE genes at 18 hpi and or 24 hpi To characterize this late signature we compared the log 2 fold change log 2 FC expression values of these 12 392 genes after infection with SARS CoV or HCoV EMC Fig 3 and www systemsvirology org There were 3 474 genes 28 expressed similarly to those in SARS CoV infected samples at the same or later times postinfection and 8 918 genes 72 expressed differently than during SARS CoV infec tion These genes were further clustered according to their expres sion pattern in HCoV EMC infected cells clusters I and III con tain genes up regulated after HCoV EMC infection at 18 to 24 hpi clusters II and IV contain down regulated genes Enrich ment in canonical pathways for each of this cluster was performed and is shown Fig 3B Genes that were up regulated by both HCoV EMC and SARS CoV cluster I though later by SARS CoV than by HCoV EMC were primarily related to viral recognition and the activation of innate immune pathways the IL 17 related pathway and activa tion of interferon regulatory factor IRF by cytosolic pattern rec ognition receptors Genes down regulated by both viruses cluster II were related to metabolism Genes specifically up regulated by HCoV EMC were statisti cally enriched in only 2 pathways cAMP mediated signaling and protein ubiquitination Because ubiquitination is involved in the innate immune antiviral response 21 this could represent an interesting mechanism to explore further Finally genes specifi cally down regulated after HCoV EMC infection were largely re lated to the antigen presentation pathway In addition the other identified canonical pathways are strongly related to lymphocyte signaling processes Together these data suggest that HCoV EMC may quickly move to interfere with elements of the adaptive im mune response in contrast to SARS CoV The antigen presentation pathway is broadly down regulatedafterHCoV EMCinfection As down regulation of the antigen presentation pathway may have important implications for the development of the adaptive response we more closely examined changes in this pathway after infection with HCoV EMC Fig 4 Twenty two genes related to major histocompati bility complex MHC class I MHC class II or antigen presenta tion were found to be down regulated after HCoV EMC infection Fig 4A while in contrast the vast majority of these genes were up regulated by SARS CoV infection after 36 hpi Fig 4B Inter estingly transcriptional regulators of MHC class I NLRC5 and MHC class II CIITA were transiently up regulated early after HCoV EMC infection at 12 and 0 hpi respectively Other genes were down regulated starting at 12 hpi Importantly both MHC class I genes HLA A B C E and G and class II genes HLA DMB DPA1 DPB1 DQA1 DRA DRB1 DRB3 DRB4 and DRB5 had decreased expression values after infection Several genes from the MHC I pathway including proteasome genes PSMB8 and PSMB9 genes from the peptide loading complex FIG 2 HCoV EMC induces more and different transcriptional changes than SARS CoV at similar times postinfection A Number of up regulated red and down regulated green differentially expressed DE genes after infection with HCoV EMC and SARS CoV compared to time matched mock infected controls The criterion used for differential expression analysis is a q value ofH110210 01 as determined by Limma s empirical Bayes moderated t test and a log 2 FC ofH110221 B Percentage of DE genes under the condition shown on the x axis that intersect with DE genes under the condition shown on the y axis To identify overlap among genes changing in the same direction up and down regulated signatures are intersected separately and the average of the two percentages is shown bya white to blue gradient 0 to 100 Host Response to HCoV EMC May June 2013 Volume 4 Issue 3 e00165 13 mbio asm org 3 mbio asm org on September 13 2015 Published by mbio asm org Downloaded from PDIA3 and TAPBP and B2M were down regulated as well as the gene for the invariant chain CD74 which belongs to the MHC II pathway Down regulation of genes within MHC I and II pathways was confirmed by quantitative reverse transcription PCR RT PCR with all 9 genes tested having significantly de creased expression in HCoV EMC samples Fig 4C Together the data show that HCoV EMC and SARS CoV induce opposite regulation of the entire antigen presentation pathway Earlyandsustainedtranscriptionalchangesmaybereverted by kinase inhibitors and glucocorticoids With the goal of iden tifying possible drugs that will modulate the host response throughout infection and from early times postinfection we fo cused on characterizing the early transcriptional changes induced by HCoV EMC that remained stable throughout the infection Among the 348 genes DE early after infection at 0 3 7 and or 12 hpi 207 59 remained dysregulated with the same pattern at later times postinfection We chose to focus on this 207 gene signature to avoid transient events that were specific to one time postinfection and to exclude genes with inconsistent expression patterns across the course of infection Expression values for these genes are displayed in Fig 5A and are available at www systems virology org This early signature was enriched in genes related to the inflammatory response with IL 17 tumor necrosis factor 2 TNFR2 related pathways and predicted activation of che motaxis of leukocytes see Table S1 in the supplemental material None of the 207 genes were DE early after SARS CoV infection 40 4 Log 2 FC ES Activation of IRF by Cytosolic Pattern Recognition Receptors Hematopoiesis from Pluripotent Stem Cells Diff Reg of Cytokine Prod in Mac and Th by IL 17A and IL 17F Role of IL 17F in Allergic Inflammatory Airway Diseases Role of IL 17A in Arthritis Ethanol Degradation II Superpathway of Cholesterol Biosynthesis Noradrenaline and Adrenaline Degradation Serine Biosynthesis Xenobiotic Metabolism Signaling Protein Ubiquitination Pathway cAMP mediated signaling Type II Diabetes Mellitus Signaling G Protein Coupled Receptor Signaling Hepatic Cholestasis Antigen Presentation Pathway Allograft Rejection Signaling Cytotoxic T Lymphocyte mediated Apoptosis of Target Cells Wnt catenin Signaling Nur77 Signaling in T Lymphocytes I 02468 II 02468 III 02468 IV 02468 B HCoV EMC SARS CoV I II III IV A 0371218 24 03712243036 4854 6072 hpi FIG 3 HCoV EMC massively dysregulates the host transcriptome at late times postinfection and triggers both similar and unique pathways compared to those induced by SARS CoV A Heatmap depicting the expression values of 12 392 genes DE after infection with HCoV EMC at late times postinfection union of DE genes at 18 and 24 hpi Genes were clustered into four main sets set I includes 1 599 genes that are significantly up regulated after infection with both HCoV EMC and SARS CoV set II includes 1 875 genes that are significantly down regulated after infection with both HCoV EMC and SARS CoV set III includes 3 922 genes that are significantly up regulated after infection with HCoV EMC but not DE with SARS CoV and set IV includes 4 996 genes that are significantly down regulated after infection with HCoV EMC but not DE with SARS CoV B For each of the four clusters the top 5 enriched canonical pathways are reported Enrichment score ES was defined as log 10 P value of enrichment Red lines depict the limit of significance PH11021 0 01 Josset et al 4 mbio asm org May June 2013 Volume 4 Issue 3 e00165 13 mbio asm org on September 13 2015 Published by mbio asm org Downloaded from however expression of 51 genes 24 5 was changed after 24 hpi This subset of 51 genes was enriched in cell viability molecules gluc