ATP1A1 enhances porcine reproductive and respiratory syndrome virus type 2 attachment and internalization.

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. IMPORTANCE: PRRSV continues to cause severe financial losses to the global swine industry. It is feasible to develop safe and effective antiviral strategies based on the initial step of viral infection, that is, the recognition of the virus by the cellular entry factors. However, the interactions between PRRSV and host factors initiating viral attachment and internalization are not fully understood yet. In this study, ATP1A1 was identified to promote both PRRSV-2 attachment and internalization through macropinocytosis and caveolae/raft-dependent endocytosis. These findings reveal an unrecognized entry mechanism of PRRSV-2 and provide novel insights for the development of antiviral drugs and vaccines against the virus.