Identification of a Natural Viral RNA Motif That Optimizes Sensing of Viral RNA by RIG-I

Abstract

Stimulation of the antiviral response depends on the sensing of viral pathogen-associated molecular patterns (PAMPs) by specialized cellular proteins. During infection with RNA viruses, 5′-di- or -triphosphates accompanying specific single or double-stranded RNA motifs trigger signaling of intracellular RIG-I-like receptors (RLRs) and initiate the antiviral response. Although these molecular signatures are present during the replication of many viruses, it is unknown whether they are sufficient for strong activation of RLRs during infection. Immunostimulatory defective viral genomes (iDVGs) from Sendai virus (SeV) are among the most potent natural viral triggers of antiviral immunity. Here we describe an RNA motif (DVG70-114) that is essential for the potent immunostimulatory activity of 5′-triphosphate-containing SeV iDVGs. DVG70-114 enhances viral sensing by the host cell independently of the long stretches of complementary RNA flanking the iDVGs, and it retains its stimulatory potential when transferred to otherwise inert viral RNA. In vitro analysis showed that DVG70-114 augments the binding of RIG-I to viral RNA and promotes enhanced RIG-I polymerization, thereby facilitating the onset of the antiviral response. Together, our results define a new natural viral PAMP enhancer motif that promotes viral recognition by RLRs and confers potent immunostimulatory activity to viral RNA.

Defective viral genomes are key drivers of the virus–host interaction

“…RIG-I is triggered by binding of 5′ di-or triphosphates present on uncapped RNA or short regions of double-stranded (ds)RNA. Phosphatase treatment suppresses the ability of in vitro-transcribed copy-back DVGs to trigger IFN production following transfection supporting the role of RIG-I in DVG sensing 91,94,96 . SeV DVGs can also associate with melanoma differentiation-associated protein 5 (MDA5) 88,94 , but the role for MDA5 in sensing other DVGs is less clear 99,100 .…”

“…Immunostimulatory DVGs can be recognized by pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs). While TLR signalling is not essential for production of type I IFNs in vitro in response to DVGs, RLR signalling is required 55,88,[94][95][96][97][98] . SeV copy-back DVGs are stronger immunostimulators than deletion DVGs 89 and are among the strongest known inducers of the antiviral response.…”

“…Structural modelling identified a 44 nucleotide (nt)-long stem-loop motif (DVG ) in a SeV DVG-546, a well-characterized and potent immunostimulatory copy-back DVG, which was absent in the SeV genome and spans the unique junction formed between the genomic break and rejoin points that form this copy-back DVG. Deletion of DVG reduces the DVG immunostimulatory activity, while introduction of the motif into an immunologically inert RNA improves its ability to induce the expression of type I IFNs and IFN-stimulated genes 94 . DVG acts in concert with the 5′-triphosphate motif to activate RIG-I and allows for enhanced RLR polymerization, a marker of activation 94 .…”

The innate immune response to RSV: Advances in our understanding of critical viral and host factors

“…Immunostimulatory DVGs (iDVGs) from Sendai virus trigger the antiviral response by stimulating strong signaling of intracellular PRRs RIG-I and MDA5 in infected cells[25, 95, 96]. This outstanding ability of stimulating the antiviral response in the presence of viral-encoded antagonists, and independent of type I IFN signaling, is driven by unique viral RNA secondary structures present in the DVGs[97]. …”

Endogenous Retroelements and the Host Innate Immune Sensors

“…RIG-I senses relatively short duplexed regions of RNA with 5′-triphosphate (5′-ppp) or diphosphate (5′-pp) groups, which are often present at the end of many viral genomic RNAs (Baum, Sachidanandam, & Garcia-Sastre, 2010; Goubau et al, 2014; Martinez-Gil et al, 2013; Pichlmair et al, 2006; Schlee et al, 2009; Xu et al, 2015). While all nascent transcripts (both cellular and viral) contain 5′-ppp, cellular RNAs normally undergo 5′-processing and therefore a removal of 5′-ppp prior to their nuclear export, which enables them to escape detection by RIG-I.…”