FIG. 4.

NS1 protein is not necessary for the interaction of vRNPs and viral mRNAs with hStau1-TAP. Cultures of HEK293T cells were transfected with a plasmid expressing hStau1-TAP protein or the TAP tag as a control and subsequently infected with the delNS1 virus strain at an MOI of 2 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), and material eluted from the IgG-Sepharose resin (Eluted) were analyzed by Western blotting with antibodies specific for PA, NP, NS1, and hStau1, as indicated on the right. (B) Total RNA was isolated from the cell extracts or 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. (C) Cultured 293T cells were cotransfected with the plasmid expressing hStau1-TAP protein or the TAP tag and the plasmids necessary to reconstitute an active vRNP in the absence of NS1 protein. TAP purification was performed 24 h posttransfection as described in Materials and Methods. Aliquots of the total extract (Input), material not bound to IgG-Sepharose resin (NBIgG), and material eluted after TEV digestion (Eluted) were analyzed by Western blotting with antibodies specific for PA, NP, NS1, and hStau1, as indicated on the right. The asterisk indicates a cross-reaction of the anti-hStau1 antibody.