Many members of the Coronaviridae family such as the middle east respiratory syndrome (MERS), severe acute respiratory syndrome (SARS), and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are known to be threats to public health. However, among all coronaviruses (CoVs), SARS-CoV-2, which is the causal agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, has been recognized as the most infectious CoV. Globally, SARS-CoV-2 has already claimed more than 3.8 million lives.
Previous research has revealed that CoVs are prone to cross-species transmission. Therefore, the collection of more information regarding animal CoVs is vital to predict future CoV outbreaks and prevent zoonotic transmission events.
Study: A Comparative Analysis of Coronavirus Nucleocapsid (N) Proteins Reveals the SADS-CoV N Protein Antagonizes IFN-β Production by Inducing Ubiquitination of RIG-I. Image Credit: Design_Cells / Shutterstock.com
What is SADS-CoV?
Researchers have recently revealed swine acute diarrhea syndrome (SADS)-CoV, which belongs to the genus Alphacoronavirus, as a novel pathogen that causes diarrhea in newborn piglets. SADS-CoV, which is also known as swine enteric alphacoronavirus (SeACoV), had reported mortality rates above 35% in southern China during a 2017 outbreak.
Apart from SADS-CoV, four more porcine CoVs have been identified to date; namely, transmissible gastroenteritis virus (TGEV), porcine hemagglutinating encephalomyelitis virus (PHEV), porcine epidemic diarrhea virus (PEDV), and porcine delta coronavirus (PDCoV). As SADS-CoV is closely related to bat CoV HKU2 strains, scientists believe this strain has emerged as a result of genetic drift or recombination occurrences between co-infecting CoVs.
Genomic studies have shown that SADS-CoV comprises a genetic sequence that consists of four structural proteins, seven independent open reading frames (ORFs) that encode sixteen non-structural proteins, and one accessory protein, all of which are similar to that which is present in many CoVs. Out of the four structural proteins, the nucleocapsid (N) protein contains a highly conserved genomic sequence that is highly expressed. The N protein plays a role in viral infection and is also involved in subgenomic ribonucleic acid (RNA) transcription, viral genome replication, and its interaction with other proteins to support virion assembly.
The immune response to SADS-CoV
Previous studies have suggested that the SADS-CoV N protein is involved in the virus’s evasion of the host’s innate immune response, which is the body’s first line of defense against harmful pathogens. Further, the type I interferon (IFN) signaling pathway plays an important role in protecting the host against viral infection, which includes primary identification of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs).
Like other RNA viruses, CoVs produce PAMPs including double-stranded RNA (dsRNA) and 5′-ppp RNA intermediates in the cytoplasm during replication. These PAMPs are then identified by host pattern recognition receptors (PRRs) such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLR). Post recognition and activation of RIG-I and/or melanoma differentiation-associated gene 5 (MDA5) lead to their interaction with caspase activation and recruitment domains (CARDs).
Subsequently, prion-like polymers are formed, which stimulates the downstream
TANK binding kinase 1 (TBK1) and inhibitor of κB kinase-ϵ (IKKϵ). The activation of TBK1 leads to the phosphorylation of interferon regulatory factor 3 (IRF3) which, in turn, promotes the production of type I IFNs. This ultimately leads to the expression of hundreds of IFN-stimulated genes (ISGs).
ISGs are expressed in an autocrine and paracrine manner in an effort to protect the host cell from viral invasion. Despite these innate defenses, viruses can often evolve to evade the host cell defenses. For example, several CoVs can inhibit host IFN responses during infection.
A recent study published in Frontiers in Immunology focuses on the roles of the SADS-CoV N protein in IFN suppression during infection. In this study, researchers compared the amino acid similarities between N proteins from various CoVs belonging to four different genera. The targets of each N protein associated with IFN signaling are also discussed. The mechanism of IFN inhibition has been determined using SADS-CoV N protein via comparative analysis.
Understanding the role of the N protein in SADS-CoV
The researchers of this study revealed that for the suppression of IFN signaling, the PAMP recognition step is a critical target for the N protein. To this end, the interaction between the SADS-CoV N protein and RIG-I triggers ubiquitination, which promotes proteasome-dependent degradation. This leads to suppression of the host’s IFN responses.
This study has also evaluated several N proteins of SADS-CoV to assess their ability to inhibit IFN response. Ultimately, the researchers found that the inhibition of this response is not dependent on the amino acid sequence similarity. For example, there is a 91.2% amino acid similarity between SARS-CoV-2 and SARS-CoV. However, in terms of the mechanisms at play for SARS-CoV-2, the N protein could inhibit the activity of the IFN promoter, which is otherwise induced by RIG-I, MAVS, TBK1, and IKKϵ, whereas SARS-CoV N protein failed to do so.
Such a result highlights the importance of tertiary structure in defining protein function. There remains a gap in current research in a complete understanding of the tertiary structure of the CoV N protein. While this may be true, data on structures of the N terminal domain (NTD) and C terminal domain (CTD) of various CoV N proteins are currently available.
The result of the current research is in line with previous reports. To this end, previous studies have shown that the N protein in PEDV provokes host IFN responses by interacting with TBK1 directly. Additionally, the N protein of SARS-CoV interacts directly or indirectly with TRIM25 and the protein activator of protein kinase R (PACT) to activate RIG-I.
The present research has also suggested that the N protein of SADS-CoV targets the initial steps of the IFN response and may directly interfere with the activation of RIG-I. This comparative analysis has also demonstrated that interference using RIG-I might be the leading method for the N protein of SADS-CoV to suppress RIG-I-like receptor (RLR) signaling. This study observed that the SADS-CoV N protein targets RIG-I to inhibit IFN-β promoter activity.