FIG 3.

TIM-1 directly binds to PRRSV demonstrated by Fc-pulldown assay (A), dot blotting (B), viral binding interference (C), and a blocking experiment (D). For the Fc-pulldown assay, the recombinant TIM-1-Fc was bound to protein A/G-beads, whereas the Fc tag served as control. The beads were then incubated with the PRRSV virions. The eluted proteins were then subjected to IB. For dot blotting, TIM-1-Fc or Fc was spotted onto nitrocellulose membranes and detected by anti-human IgG antibody or anti-PRRSV GP5 MAb. After incubation with PRRSV BJ-4 for 4 h, the membranes were eaxmined for PRRSV GP5 detection. For viral binding interference, PRRSV BJ-4 virions were incubated with TIM-1-Fc (5 μg) or Fc at 4°C for 4 h, followed by inoculation into MARC-145 cells at 4°C for 1 h. For the blocking experiment, PRRSV BJ-4 virions were pretreated with anti-PS antibody or isotype control antibody at 4°C for 4 h and then inoculated into MARC-145 cells at 4°C for 1 h. The PRRSV RNA was determined by RT-qPCR. Each experiment was performed three times independently. Statistical analysis for the RT-qPCR was carried out using the Student t test (**, P < 0.01).