Understanding Viral dsRNA-Mediated Innate Immune Responses at the Cellular Level Using a Rainbow Trout Model

Abstract

Viruses across genome types produce long dsRNA molecules during replication [viral (v-) dsRNA]. dsRNA is a potent signaling molecule and inducer of type I interferon (IFN), leading to the production of interferon-stimulated genes (ISGs), and a protective antiviral state within the cell. Research on dsRNA-induced immune responses has relied heavily on a commercially available, and biologically irrelevant dsRNA, polyinosinic:polycytidylic acid (poly I:C). Alternatively, dsRNA can be produced by in vitro transcription (ivt-) dsRNA, with a defined sequence and length. We hypothesized that ivt-dsRNA, containing legitimate viral sequence and length, would be a more appropriate proxy for v-dsRNA, compared with poly I:C. This is the first study to investigate the effects of v-dsRNA on the innate antiviral response and to compare v-dsRNA to ivt-dsRNA-induced responses in fish cells, specifically rainbow trout. Previously, class A scavenger receptors (SR-As) were found to be surface receptors for poly I:C in rainbow trout cells. In this study, ivt-dsRNA binding was blocked by poly I:C and v-dsRNA, as well as SR-A competitive ligands, suggesting all three dsRNA molecules are recognized by SR-As. Downstream innate antiviral effects were determined by measuring IFN and ISG transcript levels using qRT-PCR and antiviral assays. Similar to what has been shown previously with ivt-dsRNA, v-dsRNA was able to induce IFN and ISG transcript production between 3 and 24 h, and its effects were length dependent (i.e., longer v-dsRNA produced a stronger response). Interestingly, when v-dsRNA and ivt-dsRNA were length and sequence matched both molecules induced statistically similar IFN and ISG transcript levels, which resulted in similar antiviral states against two aquatic viruses. To pursue sequence effects further, three ivt-dsRNA molecules of the same length but different sequences (including host and viral sequences) were tested for their ability to induce IFN/ISG transcripts and an antiviral state. All three induced responses similarly. This study is the first of its kind to look at the effects v-dsRNA in fish cells as well as to compare ivt-dsRNA to v-dsRNA, and suggests that ivt-dsRNA may be a good surrogate for v-dsRNA in the study of dsRNA-induced responses and potential future antiviral therapies.

Enhancing innate antiviral immune responses in rainbow trout by double stranded RNA delivered with cationic phytoglycogen nanoparticles

“…The binding pattern was punctate, which has been found previously in vertebrate cells 31 . It has been demonstrated previously that dsRNA binds rainbow trout cells via class A scavenger receptors (SR-As) 12,32 . The present work demonstrates that SR-As bind not only free HMW poly(I:C), but also Nano-HMW NPs as well.…”

“…After labeling the dsRNA was then complexed to the cationic phytoglycogen NPs. RTgutGC cells plated into 12 well-plates (2 × 10 5 cells/mL) on glass coverslips and cultured overnight were treated with fluorescently labeled free HMW poly(I:C) or Nano-HMW (500 ng/mL) in serum free medium for 3 hr at 4 or 20 °C 12 . To examine whether the uptake of HMW poly(I:C) delivered either in soluble form or by nano-delivery differs in its entry mechanism, we compared the uptake of HMW poly(I:C) and Nano-HMW in the presence or absence of the class A scavenger receptor competitive ligand, poly I, and its non-competitive counterpart, poly C, (200 µg/mL).…”

“…The expression of antiviral response genes (IFN1, Mx1, Vig3) was detected by SYBR Green real-time PCR and quantified by the CFX Connect Real-Time PCR Detection System (Bio-Rad). The primers and PCR conditions are described previously 12,49 . The relative transcript levels of these target genes were normalized to trout β-actin and presented as fold changes over the untreated control group.…”

“…The constituent cells in these tissues have the capability to respond to PAMPs via their PRRs 15–17 , thus the induction of broad-spectrum antiviral responses on mucosal surfaces is an attractive prophylactic strategy for antiviral therapeutics 4 . PAMPs, including the toll-like receptor (TLR)3 ligand polyinosnic:polycytidylic acid [poly(I:C)], have been shown to effectively limit virus replication 12 and act as adjuvants for prophylactic vaccines 18 in aquaculture applications. However, one practical limitation of using TLR ligands to control viral infections in fish is the duration and quality of innate immune responses being induced 19 .…”

Classification and function of RNA–protein interactions

“…Through base‐pairing, RNA sequences can fold into double strands with classic secondary structures, such as hairpins or long stems with bulges. Double‐stranded RNAs (dsRNAs) play key roles in various kinds of biological activities, including mRNA transport, RNA interference (RNAi; Sharp, 1999), RNA editing, and the innate immune response (Poynter & DeWitte‐Orr, 2018). The recognition and functioning of RBPs are indispensable for these processes (Figure 2a–c).…”