Production, Isolation, and Detection of Poly IC in Extracellular Vesicles from U937 Cells.

Double-stranded RNA is produced by viruses during their replicative cycle. It is a potent immune modulator and indicator of viral infection within the body. Extracellular vesicles (EVs) are lipid-bound particles released from cells homeostatically. Recent studies have shown that a commercially available dsRNA, poly inosinic: poly cytidylic acid (poly IC), can be detected within EVs. This finding opens the door for studying EVs as (1) carriers for dsRNA and (2) indicators of viral infection. To study dsRNA-containing EVs, we must have reliable methods for producing, isolating, and detecting them. This chapter uses U937, a pro-monocytic, human myeloid leukemia cell line, as the EV producer following poly IC treatment, and an immunoblot using an anti-dsRNA antibody (J2) for detection. Two methods for isolating the EVs and two methods for isolating the RNA from these EVs are described. Together, these methods effectively produce, isolate, and detect long dsRNA from EVs.

Using long, sequence-specific dsRNA to knockdown inducible protein expression and virus production via an RNAi-like mechanism.

RNA interference (RNAi) is a powerful innate immune mechanism to knock down translation of specific proteins whose machinery is conserved from plants to mammals. The template used to determine which mRNA's translation is inhibited is dsRNA, whose origin can range from viruses (long dsRNA, ∼100-1000s bp) to host (micro(mi)RNA, ∼20mers). While miRNA-mediated RNAi is well described in vertebrates, the ability of long dsRNA to guide RNAi-mediated translation inhibition in vertebrates is controversial. Indeed, as long dsRNA is so effective at inducing type I interferons (IFNs), and IFNs down-regulate RNAi machinery, it is believed that IFN-competent cells are not capable of using long dsRNA for RNAi. In the present study the ability of long, sequence specific dsRNA to knock down both host protein expression and viral replication is investigated in IFN-competent rainbow trout cells. Before exploring RNAi effects, the optimal dsRNA concentration that would funnel into RNAi without triggering the IFN response was determined. After which, the ability of sequence specific long dsRNA to target knockdown via RNAi was evaluated in: (1) uninfected host cells using inducible luciferase gene expression and (2) host cells infected with chum salmon reovirus (CSV), frog virus 3 (FV3) or viral hemorrhagic septicemia virus genotype IVa (VHSV-IVa). Induced expression studies utilized RTG-P1, a luciferase reporter cell line, and dsRNA containing luciferase sequence (dsRNA-Luc) or a mis-matched sequence (dsRNA-GFP), and subsequent luminescence intensity was measured. Anti-CSV studies used dsRNA-CSVseg7 and dsRNA-CSVseg10 to target CSV segment 7 and CSV segment 10 respectively. Inhibition of virus replication was measured by viral titration and RT-qPCR. Taking advantage of the fact that long dsRNA can accommodate more sequences than miRNAs, the antiviral capability of dsRNA molecules containing both CSV segment 7 and segment 10 simultaneously was also measured. Target sequence appears important, as dsRNA-FV3MCP did not knock down FV3 titres, and while dsRNA-VHSV-N knocked down VHSV-IVa, dsRNA-VHSV-G and dsRNA-VHSV-M did not. This is the first study in fish to provide evidence that sequence specific long dsRNA induces potent gene expression silencing and antiviral responses in vitro via an RNAi-like mechanism instead of an IFN-dependent response.

Discovery and Use of Long dsRNA Mediated RNA Interference to Stimulate Antiviral Protection in Interferon Competent Mammalian Cells.

In invertebrate cells, RNA interference (RNAi) acts as a powerful immune defense that stimulates viral gene knockdown thereby preventing infection. With this pathway, virally produced long dsRNA (dsRNA) is cleaved into short interfering RNA (siRNA) by Dicer and loaded into the RNA-induced silencing complex (RISC) which can then destroy/disrupt complementary viral mRNA sequences. Comparatively, in mammalian cells it is believed that the type I interferon (IFN) pathway is the cornerstone of the innate antiviral response. In these cells, dsRNA acts as a potent inducer of the IFN system, which is dependent on dsRNA length, but not sequence, to stimulate an antiviral state. Although the cellular machinery for RNAi is intact and functioning in mammalian cells, its role to trigger an antiviral response using long dsRNA (dsRNAi) remains controversial. Here we show that dsRNAi is not only functional but has a significant antiviral effect in IFN competent mammalian cells. We found that pre-soaking mammalian cells with concentrations of sequence specific dsRNA too low to induce IFN production could significantly inhibit vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP), and the human coronaviruses (CoV) HCoV-229E and SARS-CoV-2 replication. This phenomenon was shown to be dependent on dsRNA length, was comparable in effect to transfected siRNAs, and could knockdown multiple sequences at once. Additionally, knockout cell lines revealed that functional Dicer was required for viral inhibition, revealing that the RNAi pathway was indeed responsible. These results provide the first evidence that soaking with gene-specific long dsRNA can generate viral knockdown in mammalian cells. We believe that this novel discovery provides an explanation as to why the mammalian lineage retained its RNAi machinery and why vertebrate viruses have evolved methods to suppress RNAi. Furthermore, demonstrating RNAi below the threshold of IFN induction has uses as a novel therapeutic platform, both antiviral and gene targeting in nature.