Numb‐Associated Kinases‐Mediated AP2M1 Activation Facilitates Porcine Reproductive and Respiratory Syndrome Virus Entry

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen that poses a considerable threat to the global swine industry, particularly with emerging variants that complicate control efforts. However, the host factors involved in PRRSV entry are not well understood. In the present study, we identified members of the numb‐associated kinase (NAK) family, specifically adaptor‐associated kinase 1 (AAK1), G‐associated kinase (GAK), and BMP‐2‐inducible kinase (BMP2K), as essential regulators of PRRSV entry utilizing both genetic and pharmacological approaches. Mechanistically, NAKs facilitate PRRSV entry by phosphorylating the adaptor protein complex 2 subunit mu‐1 (AP2M1) at threonine 156, enhancing AP2M1 activation and thereby promoting its interaction with the YxxØ motif in PRRSV glycoprotein (GP) 5 and the receptor cluster of differentiation 163 (CD163). This interaction is critical for efficient trafficking of the virions to early endosomes (EEs). Disruption of AP2M1 phosphorylation or blockade of the AP2M1–YxxØ interaction significantly impaired PRRSV internalization, indicating the potential for targeting this pathway to inhibit infection. Notably, inhibition of the NAKs–AP2M1 axis effectively reduced infection across multiple PRRSV strains, highlighting its capacity as a broad‐spectrum antiviral target. Collectively, our findings provide novel insights into PRRSV entry mechanisms and offer a promising therapeutic strategy to control emerging variants of this economically significant disease.