Hexanucleotide element of miR-29b interacts with IPO5 and mediates the protection of miR-29b against herpes simplex encephalitis in mice

Viruses have evolved sophisticated immune evasion strategies to establish persistent and latent infections. While type I interferon (IFN-I) signaling through the IFNAR–JAK–STAT axis constitutes a major antiviral defense, IFNAR-independent mechanisms also play complementary roles in restricting viral replication. Herpes simplex virus type 1 (HSV-1), a widespread DNA virus capable of lifelong latency, can exploit the host nuclear transport machinery to facilitate its replication. Here, we identify a distinct IFNAR-independent antiviral mechanism mediated by the nuclear-enriched microRNA miR-29b. We show that miR-29b, but not its closely related family member miR-29a, restricts HSV-1 replication by targeting the nuclear transport receptor Importin-5 (IPO5). Proteomic, imaging, and biochemical analyses reveal that miR-29b binds to IPO5 and disrupts IPO5-mediated nuclear import of the HSV-1 protein UL47, a key regulator of viral genome packaging. Notably, mutation of the hexanucleotide element “AGUGUU” in miR-29b by using Prime editing technology, which governs its nuclear localization, abolishes both its interaction with IPO5 and its antiviral activity in vitro and in vivo, exacerbating the progression of herpes simplex encephalitis. Our findings uncover a previously unrecognized miRNA–protein regulatory axis, in which miR-29b interferes with viral hijacking of host nuclear import pathways to selectively block viral protein trafficking, highlighting miR-29b and IPO5 as promising targets for antiviral intervention.