FIG 4.

Sialic acids on PRRSV are recognized by the NMHC-IIA–DAP12-Syk pathway to suppress PRRSV-triggered proinflammatory responses. (A) Neither PRRSV structural glycoproteins nor viral genome induces activation of the DAP12-Syk pathway. PAMs were inoculated with the same amounts (MOI = 5) of naive, UV-inactivated, and heat-inactivated PRRSV virions for 1 h. IB was performed to detect the abundance of NMHC-IIA, DAP12, and Syk, as well as phosphorylated DAP12 and Syk. (B) Heat-inactivated PRRSV virions induce DAP12-Syk activation dependent on sialic acids. The virions were treated with PNGase F, α2-3,6,8 neuraminidase or α2-3 neuraminidase S for 90 min and then were used to inoculate PAMs for 1 h. IB was used to detect phosphorylated DAP12 as well as total DAP12 and Syk. (C) NMHC-IIA interacts with PRRSV partially dependent on the sialic acids. Fc-NMHC-IIA in Fc pulldown was conducted with the same amounts of PNGase F-, α2-3,6,8 neuraminidase-, or α2-3 neuraminidase S-treated virions. IB was performed to detect PRRSV N. (D and E) IIA-C is responsible for the interaction of NMHC-IIA with PRRSV GP5. GST-tagged IIA-A, IIA-B, or II-C in GST pulldown was performed with PRRSV virions (D) or GP5 (E). Eluted proteins were subjected to IB. (F) The interaction of IIA-C and GP5 is partially dependent on the sialic acids. IIA-C–GST in GST pulldown was conducted with PNGase F- or α2-3,6,8 neuraminidase-treated GP5 followed by IB analysis. (G and H) Heat-inactivated virions antagonize LPS-triggered NF-κB activation dependent on their sialic acids. PAMs were stimulated with LPS (10 μg/ml) for 30 min in the presence of PNGase F-, α2-3,6,8 neuraminidase-, or α2-3 neuraminidase S-treated virions. IB was performed to detect the indicated proteins in NF-κB and MAPK pathways (G) and determine nuclear transportation of p65 (H). Experiments in all panels were repeated at least three times, and similar results were obtained.