Question

Human cells contain a protein that binds to the 5? triphosphate groups of RNA. Explain why this protein would be part of the defense against viral infection.

Answer 1

The ATPase retinoid acid-inducible gene (RIG)-I senses viral RNA in the cytoplasm of infected cells and subsequently activates cellular antiviral defense mechanisms. RIG-I recognizes molecular structures that discriminate viral from host RNA.

Viral infections are sensed by pattern-recognition receptors (PRRs) of the innate immune system that recognize pathogen-associated molecular patterns (PAMPs), and trigger antiviral gene programs, including the production of IFN type-I. Viral RNA serves as a PAMP and can be recognized by toll-like receptor (TLR)-3, TLR-7/8, double-stranded (ds)RNA-activated protein kinase (PKR), and the retinoid acid-inducible gene (RIG)-I-like helicase (RLH) family members RIG-I, melanoma differentiation-associated protein 5 (MDA-5), and laboratory of genetics and physiology (Lgp)2. There is evidence that RIG-I signals on infection by many RNA viruses, including important human pathogens . After ligand-mediated activation critically involving ATPase activity and the C-terminal regulatory domain (RD) RIG-I binds via its amino-terminal caspase-activation and recruitment domain (CARD) to the adaptor protein Cardif (MAVS, VISA, Ips-1) that then triggers the ?F?? and IRF signaling pathways. The exact nature of the PAMP that allows RIG-I to discriminate viral from host RNA in the cytosol is highly controversial. Kim et al. have shown that RNA produced by in vitro transcription (IVT) bearing a 5?-triphosphate end is able to trigger IFN production in cells. Thereafter, our laboratory and others have reported that an essential feature of the viral RNA ligand of RIG-I is a free 5?-triphosphate that is absent from host cytoplasmic RNA due to eukaryotic RNA metabolism. Using 5?-triphosphate RNAs produced by IVT, these studies concluded that both single-stranded (ss) and dsRNAs activate RIG-I as long as they carry the 5?-triphosphate. The RD of RIG-I has subsequently been characterized as the structural entity that binds 5?-triphosphate and, thus, aids in defining ligand specificity. However, the concept that the 5?-triphosphate modification in cytosolic RNA represents the complete PAMP recognized by RIG-I was challenged recently by several prominent studies, suggesting that (i) 3?-monophosphate RNAs, as produced by RNase L, might be RIG-I ligands; (ii) a 5?-triphosphate end is dispensable if the RNA ligand is double stranded and carries either 5?-monophosphates or is long enough ; and (iii) RIG-I ligands require uridine- or adenosine-rich sequences. These reports raise the question whether the 5?-modification with (tri) phosphate is sufficient, merely required, or in some cases dispensable for physiological RIG-I ligands. For this report, we have investigated the structural requirements to activate RIG-I-mediated antiviral signaling using defined ligands including synthetic 5?-triphosphate RNA.