Abstract

Na+-K+-ATPase alpha subunit 1 (ATP1A1) is the main functional part of the sodium pump. In addition to the well-known role in ion transport, it also acts as a signal transducer. Porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) utilizes multiple entry factors and pathways to initiate infection, posing a significant challenge to the global swine industry. However, the molecules conferring PRRSV-2 infection have not been fully characterized. Here, ATP1A1 is identified as a novel factor in PRRSV-2 attachment and internalization. ATP1A1 formed clusters in the plasma membrane very early following PRRSV-2 infection and co-internalizes with virions. Knockdown of ATP1A1 significantly suppressed PRRSV-2 infection by reducing viral attachment, and the specific chemical ligands, ouabain and PST2238, effectively reduced viral internalization without affecting viral attachment, leading to decreased viral infection. Mechanically, ATP1A1-Src signaling-dependent activation of EGFR and caveolin-1 was required for efficient PRRSV-2 uptake through macropinocytosis and caveolae/raft-mediated endocytosis. Furthermore, internalized virions were subsequently trafficked to ATP1A1/CD163-positive early endosomes, where uncoating occurs. In detail, PRRSV glycoprotein 4 (GP4), a major determinant for viral cellular tropism, was found to interact with the fourth extracellular region (ER4) of ATP1A1, dependent on its C-terminus. A synthetic ATP1A1-ER4 peptide inhibited PRRSV-2 replication by competitively reducing viral attachment and internalization in a dose-dependent manner. Most importantly, a specific nanobody targeting ATP1A1-ER4 provided broad inhibition against various PRRSV-2 lineages both in PAMs and Marc-145 cells. Collectively, these results elucidate that ATP1A1 is important for PRRSV-2 attachment and internalization, offering a potential target for the development of antiviral treatments.