FIG. 6.

Characterization of capsid RA mutants. COS cells were transfected with expression vectors encoding the RV structural proteins (24S) or the RV structural proteins containing arginine-to-alanine mutations in the capsid (24S5RA, 24S6RA, or 24S11RA). (A) Cells were biosynthetically labeled with [³⁵S]methionine-cysteine prior to lysis and immunoprecipitation with either goat anti-p32 serum (upper panel) or rabbit anticapsid serum (lower panel). Samples were subjected to SDS-PAGE and fluorography. The positions of capsid and p32 are indicated to the right of the gels. (B) Cells were labeled with H₃³³PO₄, and capsid was immunoprecipitated from lysates. Samples were subjected to SDS-PAGE and fluorography (upper panel). The relative expression levels of capsid were determined by immunoblotting of cell lysates (lower panel). (C) The relative levels of RLP- and cell-associated (Lysate) capsid from transfected cells were determined by immunoblotting. (D) Wild-type (C) and mutant capsid proteins (CS46A and C5RA) were immunoprecipitated from transfected cells, treated with phosphatase, separated by SDS-PAGE, and then transferred to nitrocellulose membranes. Membranes were incubated with ³⁵S-labeled RV genomic RNA and washed, and RNA binding to capsid was detected using a phosphorimager (upper panels). Relative capsid expression levels were determined by stripping the membranes and immunoblotting with a monoclonal antibody to capsid (lower panels). UT, untransfected cells.