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

## TL;DR

A new engineered miRNA with a 5′-triphosphate end can both activate the immune system and target PRRSV, offering a promising antiviral strategy.

## Contribution

A bifunctional 5′-PPP miRNA that combines RIG-I activation and sequence-specific viral targeting is designed and tested.

## Key findings

- 5′-PPP miRNA triggers strong interferon responses and inhibits PRRSV replication in MARC-145 cells.
- In porcine alveolar macrophages, antiviral effects mainly depend on interferon responses, not RNAi targeting.
- 5′-PPP modification enhances miRNA stability and gene-silencing activity.

## Abstract

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.