FIG 6.

Silencing RRP1B reduces RdRp binding to capped RNA. HEK293T knockdown cells were cotransfected with plasmids expressing HA-tagged PB1, PB2, and PA. At 48 h posttransfection, the cells were harvested. (A) Cell lysates were prepared using RIP lysis buffer and used for immunoprecipitation with mouse IgG or anti-m7G antibody. The cell lysates (Input) and the immunoprecipitates were resolved on SDS-PAGE and immunoblotted with anti-HA antibody. (B and C) Cell lysates were prepared using MPER buffer and used for RNA pulldown assays with desthiobiotinylated RNA-captured streptavidin beads as described in Materials and Methods. Four kinds of RNA, i.e., capped RNA, uncapped RNA, the 5′ terminus of vRNA, and poly(A), were used for the assay. The cell lysates (Input) and pulldown proteins were resolved on SDS-PAGE and immunoblotted with anti-HA antibody.