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[Preprint]. 2025 Aug 2:2025.08.02.668285. [Version 1] doi: 10.1101/2025.08.02.668285

Terminal Loop Sequences in Viral Double-Stranded RNAs Modulate RIG-I Signaling

Matthew Hackbart 1, Patrick Wang 1, Victoria Gnazzo 1, Carolina B López 1,2,*
PMCID: PMC12324521  PMID: 40766647

Abstract

Detection of foreign RNAs is a crucial activation step for innate immunity pathways in response to viral infections. Retinoic acid-inducible gene I (RIG-I) is a cytoplasmic RNA sensor that triggers type I and III interferon (IFN) expression and activates the antiviral response. The activating ligand for RIG-I has been shown to be 5’-triphosphated blunt-ended double stranded(ds) RNA, but questions remain on the molecular mechanisms for RIG-I activation during viral infections. Here we show that immune-activating copy-back viral genomes (cbVGs) contain RNA stem loops away from the 5’ end of the RNA that enhance RIG-I signaling and IFN expression. Importantly, the sequence of the terminal loops of the activating motifs impacts the strength of IFN expression. Additionally, we show that synthetic versions of these cbVG-derived stem loops trigger innate immune responses in mice demonstrating their potential as immunostimulants in vivo.

INTRODUCTION

Retinoic acid-inducible gene I (RIG-I) is an important cytoplasmic sensor of viral infections that initiate the antiviral immune response. The RIG-I-like receptor (RLR) family includes RIG-I, melanoma differentiation-associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), which are host proteins that bind to RNAs and initiate the antiviral response1,2. Binding of RLRs by foreign RNA triggers a signaling cascade through the mitochondrial antiviral signaling (MAVS) protein that ends in the expression of antiviral proteins, including type I and III interferons (IFNs)3–7. RIG-I contains a C-terminal regulatory domain that recognizes the foreign RNA and drives conformational changes in the protein helicase domain to wrap around the RNA8–13. These conformational changes also release caspase activation and recruitment domains (CARDs) that promote oligomerization of the RIG-I proteins and signaling through MAVS3,14,15. RLRs recognize unique pathogen associated molecular patterns (PAMPs) found in foreign RNA but not in the host cell RNAs.

The most well-studied of the RLR PAMPs are within the RNAs that activate RIG-I. The regulatory domain of RIG-I detects terminal 5’ triphosphates on RNA16–19 and the detection of this motif can be inhibited by methylation of the 5’ end of the RNA20,21. While ssRNA can activate RIG-I, the highest activation occurs when blunt ended dsRNA22 contains the 5’ triphosphate17. The addition of overhangs to the end of the dsRNA limits activation of RIG-I18,19. RIG-I is activated best by short dsRNA while longer synthetic dsRNAs such as poly I:C (> 500nt in length) activate MDA523–25. With these guidelines in place, minimal ligands for activating RIG-I that contain a short 10 base pair dsRNA stem with a 5’ triphosphate have been synthetically generated26,27. While these studies have provided critical data to identify the motifs that RIG-I will bind to in the RNA, in general, they are not based on RNAs found naturally during viral infections. Further work is thus needed to confirm and identify the characteristics of viral RNAs that activate RIG-I signaling during infection.

Previously, we and others determined that the RNA that most robustly activates RIG-I signaling during Sendai Virus (SeV) infection is a copy-back viral genome (cbVG)28–30. CbVGs contain complementary ends predicted to form a blunt-ended dsRNA PAMP with a 5’ triphosphate, characteristic of the canonical RIG-I ligand31,32. However, detailed folding analyses of the most prominent cbVG from SeV (cbVG 546) revealed a much more complex folding of the molecule, including a stem loop that is necessary for strong activation of the RIG-I pathway to stimulate IFN expression. This stem loop is comprised of nucleotides 70–114 of cbVG 546, and when transferred to inert RNAs, it significantly enhanced the immunostimulatory capability of the RNA33. Interestingly, the removal of stem loop 70–114 from cbVG 546 severely decreased RIG-I activation even though the cbVG still contained the 5’ triphosphate and blunt-end dsRNA.

It is unknown how SeV 70–114 enhanced RIG-I activity or whether other RIG-I stimulatory cbVGs contain stem loops similar to SeV 70–114. We therefore sought to determine if other viral cbVGs contain RNA stem loops that can transfer RIG-I stimulatory activity to otherwise inert RNAs and identify features of these RNA stem loops that are necessary for robust RIG-I activation. Here, we identified multiple sequences in cbVGs from human respiratory syncytial virus (RSV) and Nipah virus (NiV) infections that can transfer RIG-I stimulatory activity to the inert X RNA from the hepatitis C virus. Through mutation of the terminal loop sequences of the cbVG-derived RNAs, we discovered that specific nucleotides in the terminal loop of the RNA are necessary for strong activation of RIG-I signaling in cells. Lastly, in vivo administration of the cbVG-derived RNA loops activated innate immune signaling pathways, suggesting their potential use as immunostimulants for vaccines and immunotherapies.

RESULTS

RSV and NiV cbVGs stimulate interferon expression

To identify additional natural RNA stem loops that could activate antiviral innate immune pathways, we selected previously identified cbVGs from either RSV (RSV cbVG 236)34 and NiV (DI15, here called NiV cbVG 378)35. We first determined if these cbVGs can activate the IFN signaling pathway in human A549 lung cells and found that in vitro transcribed RNAs of both RSV cbVG 236 and NiV cbVG 378 could stimulate the transcription of type I (IFNB1) and type III (IFNL1) interferons at 6 hours post transfection, but not the hepatitis C virus X region (Fig 1A and 1B). This IFN activation was largely MAVS-dependent, confirming activation of the RLR signaling, similar to what we had reported for SeV cbVG 268, a shortened version of cbVG 546 (Fig 1C)33.

Figure 1. CbVG from RSV and NiV induce MAVS-dependent IFN expression.

Figure 1.

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(A+B) Quantitative PCR (qPCR) for (A) type I interferon (IFNB1) and (B) type III interferon (IFNL1) expression by A549 cells at 6 hours post transfection (hpt) with 5 pmol of HCV X RNA, SeV cbVG 268, RSV cbVG 236, or NiV cbVG 378. One-way ANOVA was performed for statistical analysis. ns: p>0.05; *: p<0.05, ***: p<0.001; ****: p<0.0001. Data are represented as fold change over Mock. (C) qPCR for IFNL1 of control or MAVS-KO A549 cells at 6 hpt with 5 pmol of SeV cbVG 268, RSV cbVG 236, or NiV cbVG 378. One-way ANOVA was performed for statistical analysis. ****: p<0.0001. Data are represented as fold change over Mock. Data reported are biological triplicates.

RSV cbVGs contain immunostimulatory stem loops

To identify which specific RSV cbVG 236 sequence confers a strong RIG-I stimulatory activity to the molecule, we first made a series of deletions in cbVG 236 that either removed internal sequences disrupting the stem loops or removed a complementary end of the cbVG (Fig 2A). We found that nucleotides 1–48 and 189–236 in the complementary ends of cbVG 236 contribute to RIG-I activation, as deletion mutants del 1–48 and 189–236 lost immunostimulatory activity (Fig 2B). Interestingly, we also found that nucleotides 49–100 are necessary for maximum IFN expression. Mutants with deletions in this range (del 49–187 and del 64–187) show reduced stimulation of the IFN response, but del 101–147 and del 137–187 induced strong IFN expression.

Figure 2. RSV cbVG sequences 1–48 and 39–68 are immunostimulatory.

Figure 2.

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(A) Indicated deletions of RSV cbVG 236 were generated by in vitro transcription then 1 pmol of IVT RNA was transfected into A549 cells. (B) Expression of IFNL1 was measured by qPCR at 6 hpt. One-way ANOVA was performed for statistical analysis. ns: p>0.05; ***: p<0.001, ****: p<0.0001. Data are represented as fold change over cbVG 236. (C) Sequences from RSV cbVG 236 were attached to X RNA then in vitro transcribed. 1 pmol of RNA was transfected into A549 cells and expression of IFNB1 and IFNL1 was measured by qPCR at 6 hpt. One-way ANOVA was performed for statistical analysis. ns: p>0.05; *: p<0.05, ****: p<0.0001. Data are represented as fold change over the X RNA control. (D) Predicted structure of RNA stem loops from RSV cbVG 236. Data reported are biological triplicates.

To determine if these RNA sequences can enhance expression of IFNs to an otherwise inert RNA molecule, we transferred the RSV cbVG sequences to the inert X RNA motif from the hepatitis C virus, ensuring that the overall structures of the final molecules were similar (Fig S2)36. We found that X-region chimeras carrying RSV cbVG sequences 1–48 or 39–68 induced the highest IFN expression when transfected into A549 cells, but RSV cbVG sequence 189–236 did not significantly enhance IFN expression in this context (Fig 2C), suggesting that additional features of the 189–236 region participate in inducing IFN expression.

To determine if specific immunostimulatory stem loops were present within the immunostimulatory sequences, we then performed in silico prediction of the RNA stem loops using RNAfold37. Since the complement ends of the cbVGs interfere with the in silico folding predictions based on minimal free energy, and it is likely that favorable secondary structures form during RNA synthesis rather than once the entire molecule has been produced, we determined all possible RNA structures on partial cbVGs lacking either the 3’ or 5’ complementary sequences. RSV cbVG 236 was predicted to form five RNA stem loops (Fig 2D and Fig S1) in the absence of 3’−5’ complementation with two of the stem loops (1–34 and 39–68) present within the immunostimulatory sequences. RNA folding of X region + RSV 1–48 predicted an immunostimulatory stem loop of sequence 1–34 similar to the full length cbVG (Fig S2).

NiV cbVGs contain immunostimulatory stem loops

Similar to RSV, we made a series of deletions to remove specific RNA sequences from NiV cbVG 378 and determined which portions of the cbVG are necessary for strong IFN expression. We found that nucleotides within the complementary ends of the cbVG were necessary for strong expression of IFNs and removal of nucleotides 325–360 also decreased IFN expression (Fig 3A and B). When attached to the X RNA, we found that while NiV 325–360 induced IFNB1 and IFNL1 expression, NiV sequences 44–78 and 85–136 induced greater IFN expression (Fig 3C). NiV cbVG 378 is predicted to have eight RNA stem loops (Fig 3D and Fig S3) with stem loops of NiV 44–78 and NiV 85–136 enhancing IFN stimulation when transferred to the inert X RNA. Unexpectedly, deletion of 44–78 or 85–136 sequences did not significantly reduce IFNL1 expression from upon RNA transfection, suggesting that the RNA folds differently in these regions of the complementary ends than what was predicted in silico.

Figure 3. NiV cbVG sequences 44–78 and 85–136 are immunostimulatory.

Figure 3.

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(A) Indicated deletions of NiV cbVG 378 were generated by in vitro transcription then 1 pmol of IVT RNA was transfected into A549 cells. (B) Expression of IFNL1 was measured by qPCR at 6 hpt. One-way ANOVA was performed for statistical analysis. ns: p>0.05; ***: p<0.001, ****: p<0.0001. Data are represented as fold change over cbVG 378. (C) Sequences from NiV cbVG 378 were attached to X RNA then in vitro transcribed. 1 pmol of IVT RNA was transfected into A549 cells and expression of IFNB1 and IFNL1 was measured by qPCR at 6 hpt.. One-way ANOVA was performed for statistical analysis. ns: p>0.05; *: p<0.05, **: p<0.01; ***: P<0.001; ****: p<0.0001. Data are represented as fold change over the X RNA control. (D) Predicted structure of RNA stem loops from NiV cbVG 378. Data reported are biological triplicates.

Terminal loop sequences are critical for IFN expression

We next investigated the characteristics conserved among high immunostimulatory RNA sequences versus lower immunostimulatory sequences (Fig 4A). The cbVG RNAs are predicted to form stem loops that are diverse in their size and shape. However, two complementary sequences that are predicted to form loops with the same structure (RSV 1–34 and RSV 203–236) exhibit different immunostimulatory capabilities (Fig 2C). We did not observe significant differences in the minimum free energy (MFE) between the stem loops, most of which had a predicted MFE between −6 and −10 kcal/mol. Upon observation, we found that the terminal loops with the higher immunostimulatory activity contain either a CAA motif or poly-A stretch at the tip of the loop (Fig 4A). In contrast, the low immunostimulatory loops are instead U-rich at the terminal loop residues, except for NiV 324–361 which contains an A-rich terminal loop.

Figure 4. Terminal loop adenines are necessary for high IFN expression.

Figure 4.

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(A) Predicted RNA structures of high immunostimulatory and low immunostimulatory stem loops derived from cbVGs. (B-E) Indicated mutations were generated for cbVG sequences attached to the X RNA. The following sequences were mutated: (B) SeV 70–114, (C) RSV 1–48, (D) NiV 85–136, and (E) NiV 324–361. RNAs were in vitro transcribed and 1 pmol of IVT RNA was transfected into A549 cells. Expression of IFNL1 was measured by qPCR at 6 hpt. One-way ANOVAs were performed for statistical analysis. ns: p>0.05; *: p<0.05, **: p<0.01; ***: P<0.001; ****: p<0.0001. Data are represented as fold change over the wild-type stem loops. Data reported are biological triplicates.

We then hypothesized that the terminal loop adenines played a key role in triggering RIG-I signaling. To test this hypothesis, we mutated the terminal loops of the X-RNA attached sequences and tested their ability to induce IFN expression. Mutation of the terminal loop sequence did not alter the overall structure of the stem loops (Fig S4–7). For X + SeV 70–114, we found that mutation of the A89 or A90 terminal residues greatly reduced IFN activation by the RNA construct, however mutation of the C88 residue did not (Fig 4B). Mutations in X + RSV 1–48 showed that A18, but not C17 or A19, was necessary for full IFN activation (Fig 4C). Additionally, mutation of X + NiV 85–136 showed that both terminal adenines, A111 and A112, were necessary for IFN signaling (Fig 4D). However, this trend was not observed for the low immunostimulatory X + NiV 324–361, which did not show a reduction in IFN stimulation upon mutation of the terminal adenines (Fig 3E). These data suggest that terminal adenines are critical to trigger robust IFN expression and that additional sequence-specific factors impact their stimulatory activity.

RSV 1–48 activates innate immune signaling in vivo

Previously, we found that the SeV cbVG 546 stem loop 70–114 activates a strong type I immune responses in mice when administered alone or within a lipid nanoparticle (LNP)38–40. Here, we aimed to determine whether RSV sequence 1–48 similarly activates innate sensors and induces an immune response in vivo. To ensure the RNA constructs were delivered intracellularly to trigger the RIG-I pathway, we packaged the X + RSV 1–48 and the control X + RSV 189–236 chimera constructs into LNPs (Fig 5A) and confirmed the homogeneity of the formulations before in vivo administration (Fig 5B). These formulations were then administered subcutaneously in the footpad of C57BL/6 mice. After 16 hours, we found that LNPs containing X + RSV 1–48 induced significantly higher expression of IFNB1, MX1, IL1B, and CXCL10 compared to empty LNPs and X + RSV 189–236 (Fig 5C). These data demonstrate that the RSV cbVG-derived 1–48 sequence retains its immunostimulatory activity in vivo when packaged into LNPs and together with data from SeV 70–114 demonstrate that small RNA motifs with terminal loop adenines can serve as potent immunostimulators in mice.

Figure 5. X + RSV 1–48 activates innate immune signaling in mice.

Figure 5.

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(A) Schematic of lipid nanoparticles (LNPs) containing X + RSV 1–48 (X 1–48) or X + RSV 189–236 (X 189–236). (B) Dynamic light scattering measurement of LNP sizes of empty LNPs, X 1–48, and X 189–236. (C) LNPs were injected subcutaneously into the footpad of mice. Sixteen hours post injection, RNA was collected and the RNA expression of IFNB1, MX1, CXCL10, and IL1B was quantified by qPCR. One-way ANOVAs were performed for statistical analysis. ns: p>0.05; **: p<0.01; ***: P<0.001; ****: p<0.0001. Data reported are 5 individual mice.

DISCUSSION

We have identified multiple RNA stem loops derived from cbVGs of RSV and NiV that can transfer robust IFN stimulatory capabilities to an inert RNA. One unique feature of the higher immunostimulatory RNA stem loops is a richness for adenines at the tip of the loop. When these sequences were mutated, the mutant RNAs stimulated significantly less IFN upon transfection into cells. Upon packaging into lipid nanoparticles, the RSV cbVG-derived stem loop was able to activate innate immune signaling in mice.

While the enhanced stimulation appears to occur with adenine-rich sequences, we also observe that a stem loop with 5 adenines (NiV 325–360) did not have decreased IFN activation when the adenines were mutated (Fig. 4E). Since an adenine-rich loop does not always enhance IFN expression, more work is necessary to determine the exact motif or motifs that make up an optimal terminal loop PAMP. Some possibilities are that sequences in the stems of the RNA interact with the loop to alter binding or that host proteins are recognizing a specific RNA motif that is more than adenines.

One major question that remains is what step of the RIG-I pathway is impacted by these terminal adenine-rich sequence. We propose the following possibilities for how the terminal loop sequences may alter IFN expression: 1) RIG-I is heavily modulated by phosphorylation and ubiquitination by host proteins41, so the terminal loop may recruit or inhibit additional host factors from interacting and altering RIG-I function in cells. 2) MAVS has recently been shown to interact with host RNAs42. The terminal sequence may stabilize RIG-I binding to MAVS through RNA/MAVS interactions and enhance downstream signaling to boost IFN expression. 3) The terminal loop adenines may be prone to RNA modifications such as N6 methylation to generate N6 methyladenosine (m6A)43. m6A RNA modification can alter the stability of the RNA in a cell and modulate the binding of RNA binding proteins. 4) Other unknown host RNA-binding proteins support robust RIG-I signaling. Further work is necessary to parse out what interactions occur with this terminal loop sequence and why adenine-rich sequences enhance IFN expression from the RIG-I pathway.

With the advent of mRNA vaccines and the usage of synthetic RNAs as therapeutics, understanding how RNAs can activate innate immunity is crucial to optimizing RNA-based adjuvants and treatments. Synthetic RNAs can act as immune adjuvants during vaccination27,38–40,44,45, and the addition of immunostimulatory stem loops to mRNA vaccines may bypass the need for additional adjuvants such as alum. Although additional work is necessary to assess the immunostimulatory properties of RNA stem loops in vivo, our discovery that the terminal loop sequence modulates immune activation can lead to the development of strategies to tailor the immune response not only based on dosage of stimulatory RNA, but on the sequence of attached RNA stem loops.

MATERIALS AND METHODS

Cells and Reagents.

A549 human type II alveolar cells (ATCC, CCL-185) and MAVS-KO A549 cells (gifted from S. Weiss) were cultured at 37°C and 5% CO2 with Dulbecco’s modified Eagle’s media (Thermofisher, 11995065) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-Glutamine, and 50 mg/mL gentamicin. All cell lines were treated with mycoplasma removal agent (MP Biomedicals, 093050044) and routinely tested for mycoplasma before use.

In Vitro Transcription of RNA.

RNAs were cloned into a pSL1180-T7 plasmid that contains T7 promoter at the 5’ end of the RNA sequence and hepatitis D virus ribozyme after the RNA sequence. In vitro transcription was performed with the Megascript T7 Transcription Kit (Thermofisher, AM1334) and RNA was isolated with LiCl precipitation according to manufacturer’s protocols. Purified RNA was measured by Qubit and tested for quality by Bioanalyzer (Agilent). Full protocol available Protocols.io: dx.doi.org/10.17504/protocols.io.3byl49qprgo5/v2

RT-PCR and qPCR.

A549 cells were transfected with 5 pmol of indicated RNA and Lipofectamine 2000 (Thermofisher, 11668027). After 6 hours, RNA was isolated with Trizol (Thermofisher, 15596026). cDNA was generated with random hexamers using the High-Capacity RNA to cDNA kit (Thermofisher, 4387406) according to the manufacturer’s protocols. For qPCR quantification, cDNA was quantified with Power SYBR Green Mix (Thermofisher, 4367660). IFNL1, IFNB1, IL1B, MX1, and CXCL10 were normalized to a house keeping index calculated from ACTB and GAPDH genes. Sequences of primers are given in Table S1. Full protocol available at Protocols.io: dx.doi.org/10.17504/protocols.io.8epv5rxw6g1b/v1

Mice

C57BL/6 mice (The Jackson Laboratory) were bred in house. All mice were sex and age matched. Both male and female mice were included in the experiments.

Lipid Nanoparticle formulation

X + RSV 1–48 and X + RSV 189–236 were encapsulated in the GenVoy Ionizable Lipid Mix (ILM) using a NanoAssemblr Ignite machine (Precision Nanosystems) following the manufacturer’s instructions. Empty LNPs were performed and used as controls. Encapsulation efficiency and RNA concentration were tested by Ribogreen Assay (ThermoFisher). Final particle size was measure by Dynamic Light Scattering. Full protocol available at Protocols.io:dx.doi.org/10.17504/protocols.io.36wgqnn4kgk5/v1

Mice immunization

Mice were anesthetized with isoflurane and injected subcutaneously (s.c.) into a rear footpad. For immune response studies mice were inoculated with 0.5 μg X 1–48; 0.5 μg × 189–236; Empty LNPs or PBS at a final volume of 30 μl per dose. RNA was extracted from footpad 16 hours after inoculation using TRIzol (Ambion Inc.).

In Silico RNA Folding Prediction.

All RNA folding predictions were performed using the University of Vienna RNAfold website with the ViennaRNA Package (Version 2.6.3). Structures shown are the minimum free energy structures. All sequences used for folding are given in Table S2. Website: http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi

Statistical Analysis and Reproducibility

All statistics were calculated using GraphPad Prism. Version 9. Specific tests and significance values are indicated in each figure legend. Data reported are biological triplicates unless otherwise stated.

ACKNOWLEDGEMENTS

The authors acknowledge the use of the Chemical and Environmental Analysis Facility at Washington University in Saint Louis for instrumentation and analytical support.

Funding:

NIH T32 HL007317-44 (MH)

NIH T32 AI007163-45 (MH)

NIH A137062 (CBL)

WashU BJC Investigator Program (CBL)

Funding Statement

NIH T32 HL007317-44 (MH)

NIH T32 AI007163-45 (MH)

NIH A137062 (CBL)

WashU BJC Investigator Program (CBL)

Footnotes

Ethics statement

All described studies adhered to the Guide for the Care and Use of Laboratory Animal of the National Institute of Health. Institutional Animal Care and Use Committee, Washington University in St. Louis approved protocol 23–0083.

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