RIG-I Activation by a Designer Short RNA Ligand Protects Human Immune Cells against Dengue Virus Infection without Causing Cytotoxicity

This study characterized a designer short hairpin RNA ligand, 3p10LG9, as a potent activator of the cytoplasmic RNA sensor RIG-I and evaluated its antiviral efficacy against dengue virus (DENV) infection in human cell lines and primary human skin antigen-presenting cells. The authors engineered structural modifications into a 5′-triphosphorylated double-stranded RNA hairpin and demonstrated that insertion of an additional guanine nucleotide created a kinked RNA structure that significantly enhanced type I interferon (IFN) induction compared with the parental construct 3p10L. Transfection of 3p10LG9 into U937-DC-SIGN monocytic cells and A549 epithelial cells induced strong IFN-stimulated signaling and inhibited DENV-2 infection in a dose-dependent manner. Mechanistic analyses showed that antiviral activity was highly dependent on RIG-I and downstream type I IFN signaling. RIG-I overexpression enhanced IFN activation, whereas MDA5 deficiency had minimal effect, confirming selective activation of the RIG-I pathway. In contrast, CRISPR-mediated RIG-I knockout abolished interferon-stimulated gene induction and antiviral activity, while IFNAR blockade reversed DENV inhibition. The study further demonstrated efficient uptake and innate immune activation of 3p10LG9 in ex vivo primary human skin dendritic cell subsets, including CD11c⁺ dermal dendritic cells, CD14⁺ dermal dendritic cells, and Langerhans cells. Transcriptomic analyses revealed robust upregulation of interferon-stimulated and antiviral response genes following 3p10LG9 treatment. Compared with poly(I·C), 3p10LG9 induced stronger antiviral transcriptional responses in several skin antigen-presenting cell populations. Functional assays showed that prophylactic treatment efficiently suppressed DENV replication with substantially lower EC50 values than the parental construct, whereas therapeutic administration after infection produced more modest effects. Overall, the study demonstrated that optimized minimal RIG-I agonist RNAs can induce potent antiviral innate immune responses with limited cytotoxicity, supporting their potential as prophylactic or therapeutic antiviral agents against dengue virus and related viral infections.
(TMR)