Cytokine systems approach demonstrates differences in innate and pro-inflammatory host responses between genetically distinct MERS-CoV isolates

Selinger, Christian; Tisoncik-Go, Jennifer; Menachery, Vineet D; Agnihothram, Sudhakar; Law, G Lynn; Chang, Jean; Kelly, Sara M; Sova, Pavel; Baric, Ralph S; Katze, Michael G

To further investigate potential regulators mediating the gene expression contrasts between MERS-CoV Eng 1 and MERS-CoV SA 1 at the late time points, we performed an Upstream Regulator Analysis in Ingenuity Pathway Analysis (IPA) that identifies upstream transcriptional regulators based on the observed gene expression changes in the experimental data set and compiled knowledge of reported relationships between regulators and their known target genes within the Ingenuity Pathway Knowledge Base. The NUPR1 gene encoding a transcription factor known as stress-activated nuclear protein 1 had a predicted inhibition (z-score of -2.373) for MERS-CoV SA 1 and predicted activation (z-score of 3.226) for MERS-CoV Eng 1 at 18 hpi (Table 3 ). This LPS-induced transcription factor is an important regulator of apoptosis, cell proliferation and autophagy [24] [25] [26] . Differentially expressed genes between MERS-CoV SA 1 and MERS-CoV Eng 1 that are binding partners and downstream targets, such as EP300 and TESK1, shown in Additional file 3: Figure S1 , were downregulated in MERS-CoV SA 1 virusinfected cells at 18 and 24 hpi and up-regulated in MERS-CoV Eng 1 virus-infected cells at these late time points, corroborating the predicted NUPR1 regulator status in response to these two MERS-CoV strains. We also identified STAT3 as a predicted upstream regulator at 24 hpi in response to MERS-CoV Eng 1 (z-score = 2.559), with up-regulation of downstream target genes including BCL6, SOCS3, BCL3, IRF7 and PML ( Figure 5 , Table 3 ). Through a separate binding motif prediction analysis using JASPAR and PSCAN, we confirmed the STAT3 prediction based on enrichment of STAT3 binding sequences in DE genes at the late time points (Table 3) . Conversely, MERS-CoV SA 1 infection resulted in downregulation of these same target genes, suggesting a predicted inhibition or delay in STAT3 regulator activity, though STAT3 did not reach a statistically significant z-score (z-score = 0.364). Among the STAT3 target genes BCL3 had the highest contrasting expression between the viruses. The BCL3 gene was highly up-regulated in response to MERS-CoV Eng 1 (FC = 1.5) and downregulated in response to MERS-CoV SA 1 (FC = -1.66) at 18 hpi. qRT-PCR analysis of BCL3 mRNA expression, and several additional STAT3 target genes, including BCL6, PML, and IRF7, were in agreement with the microarray findings (Additional file 4: Figure S2 ). Among these genes, IRF7 plays an important role in the cellular antiviral response and Faure and colleagues reported high levels of IRF7 gene expression in BAL cells from a patient that recovered from MERS infection [12] .