FIG. 2.

Association of viral proteins and RNAs with the recombinant hStau1-TAP protein in infected cells. Cultures of HEK293T cells were transfected with a plasmid expressing the hStau1-TAP protein or the TAP tag as a control and subsequently infected with WSN virus at an MOI of 5 PFU/cell. At 6 h postinfection, total cell extracts were prepared and used for TAP purification as indicated in Materials and Methods. (A) Aliquots of the total extract (Input), material not bound to IgG-Sepharose resin (NBIgG), material not bound to calmodulin resin (NBCa), and material eluted from the calmodulin resin (Eluted) were analyzed by Western blotting with antibodies specific for PB1, PA, NP, NS1, and hStau1, as indicated on the right. The asterisks on the left indicate the band of hStau1-TAP, which cross-reacts with rabbit antisera. (B) Total RNA was isolated from the cell extracts of the eluted fractions, and RT-PCR was performed with primers specific for M1 and NP RNAs of positive (c/m) or negative (v) polarity. Tenfold serial dilutions of the RNAs were analyzed until the PCR signal for the TAP control was negligible. As a negative control, the primers were omitted in the RT reaction step (−) but were included in the PCR step.