Entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis

Porcine alveolar macrophages (AM Phi) are the dominant cell type that suppo rts the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in vivo and in vitro, In order to determine the characteristics of the virus-receptor interaction, the attachment of PRRSV to cells was examin ed by using biotinylated virus in a series of flow cytometric assays. PRRSV bound specifically to AM Phi, in a dose-dependent manner. Binding of PRRSV to AM Phi increased gradually and reached a maximum within 60 min at 4 OC. By confocal microscopy, it was shown that different degrees of PRRSV bindi ng exist and that entry is by endocytosis, Virus uptake in vesicles is a cl athrin-dependent process, as it was blocked by the addition of cytochalasin D and co-localization of PRRSV and clathrin was found. Furthermore, by the use of two weak bases, NH,CI and chloroquine, it was demonstrated that PRR SV uses a low pH-dependent entry pathway. In the presence of these reagents , input virions accumulated in large vacuoles, indicating that uncoating wa s prevented. These results indicate that PRRSV entry into AM Phi involves a ttachment to a specific virus receptor(s) followed by a process of endocyto sis, by which virions are taken into the cell within vesicles by a clathrin -dependent pathway. A subsequent drop in pH is required for proper virus re plication.