Synergistic Inhibition of Porcine Reproductive and Respiratory Syndrome Virus by a Bifunctional 5'-PPP miRNA Combining RIG-I Activation with Sequence-Specific Viral Targeting.

The immunosuppressive nature of porcine reproductive and respiratory syndrome virus (PRRSV) remains the central obstacle to its effective control. Conventional microRNA (miRNA)-based antiviral approaches are limited by their modest potency and the high risk of viral escape. Here, we rationally designed an engineered miRNA carrying a 5'-triphosphate (5'-PPP) terminus that integrates RIG-I-driven innate immune activation and sequence-specific gene silencing within a single molecule. In vitro-transcribed 5'-PPP miRNAs are efficiently recognized by the pattern-recognition receptor RIG-I, triggering a robust type I interferon response that counteracts PRRSV-induced immunosuppression. In MARC-145 cells, one such construct, 5'-PPP BZL-sRNA-20, potently inhibited PRRSV replication through the synergistic action of immune activation and gene silencing. However, in porcine alveolar macrophages (PAMs)-the natural host cells for PRRSV-the antiviral effect depended primarily on 5'-PPP-induced interferon responses, with the targeting sequence providing limited or context-dependent benefits. Dual-luciferase assays confirmed that the gene-silencing activity depends on 5'-PPP modification, which enhances the stability of BZL-sRNA-20. This bifunctional strategy establishes an "immune activation plus targeting" paradigm by simultaneously acting as a RIG-I ligand that triggers broad antiviral responses and specifically cleaves viral RNA via direct base-pairing to conserved regions of the PRRSV genome. These findings reveal the potential of engineered 5'-PPP miRNAs as immunomodulatory antiviral agents, while highlighting that the contribution of RNAi targeting varies depending on the cellular context.