Interaction of recombinant influenza A virus nucleoprotein with host cellular nucleolin.

<p>A549 cells were transiently transfected with the recombinant viral NP and mock transfected with the empty vector for co-immunoprecipitation and co-localization studies. Cell lysates prepared at 24hrs post transfection were co-immunoprecipitated with anti-Myc antibody and immune complexes were subjected to immunoblotting using anti-nucleolin antibody. Another transfection experiment was carried out and recovered cell lysates were immunoprecipitated with anti-nucleolin antibody followed by immunoblotting with anti-Myc antibody in a reverse order. Further, cells grown on coverslips were transfected with the recombinant viral NP or the empty vector. Cells were fixed at 24hrs post transfection and subjected to double IFA staining with anti-viral NP (mouse) and anti-nucleolin (rabbit) antibodies. Secondary antibodies used were anti-mouse antibody tagged with fluorophore FITC (green) and anti-rabbit antibody tagged with TRITC (red). [a] Co-precipitation of 110kDa nucleolin protein with recombinant viral NP in A549 cell lysate [b] Expression of viral NP, endogenous nucleolin in transfected cells [c] Co-precipitation (reverse IP) of recombinant viral NP of ~56kDa with nucleolin [d] Expression of viral NP and endogenous nucleolin in the corresponding cell lysates [e] Co-localization of host nucleolin and viral NP (PCC: 0.56 and MOC: 0.82). Nucleus is stained with Hoechst.