The 3′ UTR Pseudoknot-Containing Region of Arteriviruses Acts as a PAMP to Activate RIG-I- and TLR8-Dependent Type I Interferon Responses

Porcine reproductive and respiratory syndrome virus (PRRSV) and other members of the Arteriviridae family cause substantial economic losses in livestock. However, their pathogen-associated molecular patterns (PAMPs) remain poorly characterized. Here, we investigated whether the 3' untranslated region (UTR) pseudoknot structures of Arteriviruses act as PAMPs and explored the underlying innate immune recognition mechanisms. Bioinformatics predictions (RNAalifold, RNArtist, and RNAComposer) revealed that the 3' UTR pseudoknot structures are conserved across all four Arteriviruses. In vitro transcribed pseudoknot RNAs significantly upregulated the mRNA expression of type I interferons (IFNα/β) and interferon-stimulated genes (protein kinase R, PKR; interferon-stimulated gene 15, ISG15; interferon-stimulated gene 56, ISG56) in Marc145 cells. Screening of RNA-sensing pattern recognition receptors (retinoic acid-inducible gene I, RIG-I; melanoma differentiation-associated protein 5, MDA5; Toll-like receptor 3/7/8/9, TLR3/7/8/9) in pseudoknot RNA-stimulated cells showed that RIG-I and TLR8 were significantly upregulated, whereas MDA5, TLR3, TLR7, and TLR9 were not. Short hairpin RNA (shRNA)-mediated knockdown of RIG-I or TLR8 significantly reduced pseudoknot-induced expression of IFNβ, PKR, and ISG56, confirming that both receptors are functionally required for this response. Upregulation of myeloid differentiation primary response 88 (MyD88), interferon regulatory factor 7 (IRF7), and signal transducer and activator of transcription 2 (STAT2) suggested that IFNα/β and downstream interferon-stimulated genes (ISGs) were induced via the TLR8-MyD88-IRF7 and RIG-I-IRF7 signaling pathways. Moreover, small-angle X-ray scattering (SAXS) analysis of the PRRSV pseudoknot RNA complexed with the porcine RIG-I regulatory domain was consistent with the formation of a complex between the pseudoknot RNA and RIG-I. In conclusion, this study identified the 3' UTR pseudoknot-containing region of Arteriviruses as a conserved PAMP that activates RIG-I- and TLR8-dependent type I IFN responses, providing a mechanistic basis and potential targets for antiviral therapy and vaccine adjuvant development against Arterivirus infections.