FIG. 8.

The VP35 IID is sufficient to interact with NP, and this interaction is impaired by either specific point mutations or the presence of dsRNA. (A) MBP or MBP-VP35 IID was added to NP-transfected cell lysates. MBP was immobilized by amylose resin, and proteins were detected with anti-MBP (α-MBP) or anti-NP (α-NP) antibodies. (B) MBP alone, MBP-VP35 IID WT, or mutants with point mutations were added to lysates containing EBOV NP. MBP was precipitated using amylose resin and was detected with NP and MBP antibodies. (C) MBP, WT MPB-VP35 IID, or MBP-VP35 IID mutants (R312A, K322A, or F239A) were added to NP-transfected lysates in the absence or presence of poly(I:C). The final concentration of each MBP was 2.4 μM, and poly(I:C) was present at either 16 or 98 nM. MBPs were pulled down, and both MBP and NP were detected in the same way as described for panels A and B. The input whole-cell extract lane (WCE) expressing NP represents 4% of the total lysate used for each group, while the MBP pull-down lane represents 15% of the total pull down.