Figure 2

RNA-binding activity of CD151 in vitro and in vivo. (A) In vito RNA-binding activity of CD151 was demonstrated by Immunoprecipitation/North-Western blot analysis. BHK-21 and MARC-145 cells were transfected with pBK-CMV plasmid expressing CD 151 as a β-galactosidase fusion protein. The cell lysates were immunoprecipitated with anti-CD151 MAb (A1) and anti-β-galactosidase MAb (A2). In A1, lane1, MARC-145 cytoplasmic protein lysate (without immunoprecipitation); lanes 2, transfected MARC-145; lane 3, transfected BHK-21; lane 4, untransfected MARC-145; lane 5, untransfected BHK-21. In A (2), lane1, MARC-145 cytoplasmic protein lysate (without immunoprecipitation); lane 2, untransfected BHK-21; lane 3, transfected BHK-21; lane 4, transfected MARC-145; lane 5, untransfected MARC-145. FIG 2B, gel shift assay demonstrating the interaction of CD151 protein with the PRRSV 3'UTR RNA. MARC cell lysate was immunoprecipitated with CD151 antibody (lanes 1&3) and the complex was incubated radiolabelled PRRSV 3' UTR RNA. Addition of unlabelled RNA (lane 3) prevented the formation of complex, while the radiolabelled RNA did not interact with the CD151 antibody (lane 3). FIG 2C, In vivo RNA-binding activity of CD151 was demonstrated by immunoprecipitation/RT-PCR assay (149 bp amplicon). PRRSV-infected or uninfected MARC-145 cell lysates were immunoprecipitated with anti-CD151 MAb or a negative control MAb (wasp, Cotesia folepis MAb), and RT-PCR was performed using PRRSV 3' UTR RNA-specific primers for RNAs extracted from the immunocomplexes. M, 123 bp ladder; lane 1, negative PCR control; lane 2, PRRSV-uninfected/CD151 MAb-immunoprecipitated; lane 3, PRRSV-infected/wasp MAb-immunoprecipitated; lane 4, PRRSV-infected/CD151 MAb-immunoprecipitated (without UV cross-linking); lane 5, PRRSV-infected/CD151 MAb-immunoprecipitated (UV cross-linked for 15 min); lane 6, PRRSV-infected/CD151 MAb-immunoprecipitated (UV cross-linked for 30 min); lane 7, PRRSV-infected/CD151 MAb-immunoprecipitated (UV cross-linked for 45 min).