FIG 4.

Mutations to RIG-I enable direct V protein targeting. (A) RIG-I signaling assay similar to that described for Fig. 3A, but using a broader range of measles virus or PIV5 V protein expression (4, 20, 100, 500 ng). Immunoblots below demonstrate protein expression levels in representative lysates. (B) FLAG-tagged RIG-I or variants were coexpressed in HEK293T cells with HA-tagged V proteins from PIV5 and measles virus. Cell lysates were prepared and analyzed as described for Fig. 1B. (C) Combinatorial analysis of RIG-I mutations required for PIV5 V interaction. RIG-I mutants and PIV5 V were coexpressed and prepared as described above. (D) The RIG-I L714R E716G mutant is biologically active and suppressed by V proteins. Luciferase assays were carried out similar to those described for Fig. 4A, but using the RIG-I L714R E716G mutant in the presence and absence of measles virus or PIV5 V protein titration (4 ng, 20 ng, 100 ng, and 500 ng). Student's t test indicated as follows: N.S. (not significant), P > 0.05; *, P < 0.05.