Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Suppresses Type I and Type III Interferon Induction by Targeting RIG-I Signaling.

Type I and type III interferons (IFNs) are the frontline of antiviral defense mechanisms that trigger hundreds of downstream antiviral genes. In this study, we observed that MERS-CoV nucleocapsid (N) protein suppresses type I and type III IFN gene expression. The N protein suppresses Sendai virus-induced IFN-ß and IFN-λ1 by reducing their promoter activity and mRNA levels, as well as downstream IFN-stimulated genes (ISGs). Retinoic acid-inducible gene I (RIG-I) is known to recognize viral RNA and induce IFN expression through tripartite motif-containing protein 25 (TRIM25)-mediated ubiquitination of RIG-I caspase activation and recruitment domains (CARDs). We discovered that MERS-CoV N protein suppresses RIG-I-CARD-induced, but not MDA5-CARD-induced, IFN-ß and IFN-λ1 promoter activity. By interacting with TRIM25, N protein impedes RIG-I ubiquitination and activation and inhibits the phosphorylation of transcription factors IFN-regulatory factor 3 (IRF3) and NF-κB that are known to be important for IFN gene activation. By employing a recombinant Sindbis virus-EGFP replication system, we showed that viral N protein downregulated the production of not only IFN mRNA but also bioactive IFN proteins. Taken together, MERS-CoV N protein functions as an IFN antagonist. It suppresses RIG-I-induced type I and type III IFN production by interfering with TRIM25-mediated RIG-I ubiquitination. Our study sheds light on the pathogenic mechanism of how MERS-CoV causes disease.IMPORTANCE MERS-CoV causes death of about 35% of patients. Published studies showed that some coronaviruses are capable of suppressing interferon (IFN) expression in the early phase of infection and MERS-CoV proteins can modulate host immune response. In this study, we demonstrated that MERS-CoV nucleocapsid (N) protein suppresses the production of both type I and type III IFNs via sequestering TRIM25, an E3 ubiquitin ligase that is essential for activating the RIG-I signaling pathway. Ectopic expression of TRIM25 rescues the suppressive effect of the N protein. In addition, the C-terminal domain of the viral N protein plays a pivotal role in the suppression of IFN-ß promoter activity. Our findings reveal how MERS-CoV evades innate immunity and provide insights into the interplay between host immune response and viral pathogenicity.

Nucleocapsid protein-dependent assembly of the RNA packaging signal of Middle East respiratory syndrome coronavirus.

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) consists of a positive-sense, single-stranded RNA genome and four structural proteins: the spike, envelope, membrane, and nucleocapsid protein. The assembly of the viral genome into virus particles involves viral structural proteins and is believed to be mediated through recognition of specific sequences and RNA structures of the viral genome. METHODS AND RESULTS: A culture system for the production of MERS coronavirus-like particles (MERS VLPs) was determined and established by electron microscopy and the detection of coexpressed viral structural proteins. Using the VLP system, a 258-nucleotide RNA fragment, which spans nucleotides 19,712 to 19,969 of the MERS-CoV genome (designated PS258(19712-19969)ME), was identified to function as a packaging signal. Assembly of the RNA packaging signal into MERS VLPs is dependent on the viral nucleocapsid protein. In addition, a 45-nucleotide stable stem-loop substructure of the PS258(19712-19969)ME interacted with both the N-terminal domain and the C-terminal domain of the viral nucleocapsid protein. Furthermore, a functional SARS-CoV RNA packaging signal failed to assemble into the MERS VLPs, which indicated virus-specific assembly of the RNA genome. CONCLUSIONS: A MERS-oV RNA packaging signal was identified by the detection of GFP expression following an incubation of MERS VLPs carrying the heterologous mRNA GFP-PS258(19712-19969)ME with virus permissive Huh7 cells. The MERS VLP system could help us in understanding virus infection and morphogenesis.