FIG. 4.

EBOV VP35 interacts with TRBP and PACT: model for VP35. VP35 interacts with the Dicer/PKR partners TRBP and PACT but not with Dicer. (A) HEK293 cells were transfected with His-tagged pVP35 (pVP35-H) alone or with pDicer-F, pTRBP-F, or pPACT-F. Total cellular extracts were immunoprecipitated with anti-FLAG. Dicer, TRBP, and PACT immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting using an anti-His antibody (α-FLAG IP). pluc-F was used as a negative control. Input lanes contain 10% of the total cell lysates and were detected with anti-His (Input). This experiment was performed three times. (B) Binding of dsRBD/IFN VP35 K309A and R312A mutants to TRBP/PACT. HEK293 cells were transfected with pVP35(K309A)- or pVP35(R312A)-His alone or with pDicer-F, pTRBP-F, or pPACT-F. Total cellular extracts were immunoprecipitated with anti-FLAG. Coimmunoprecipitation of VP35 mutants was detected using anti-His antibody (α-FLAG IP). pluc-F was used as a negative control. Input lanes contain 10% of the total cell lysates and were detected with anti-His (Input). (C) Molecular mechanism of VP35. By binding to Dicer/PKR partners, VP35 affects both RNAi and PKR pathways that require both TRBP and PACT. Domains of PACT and TRBP are shown as domains A, B, and C. TRBP-PACT heterodimer, domains A and B; Dicer (or PKR) binding, domain C. PACT, cyan oval; TRBP, yellow oval; Dicer, green oval; PKR, red triangle.