Figure 4: FMRP interacts with NP in an RNA-dependent manner and stimulates viral RNP assembly and viral replication.

(a) NP co-purifies with FMRP. The control vector, TAP-tagged PA, PB1, PB2 or NP expression constructs were individually transfected into 293T cells along with an FMRP-Flag plasmid. After 36 h, cells were lysed followed by TAP purification and western blot analyses. (b) FMRP associates with NP in WSN-infected cells. 293T cells were left untransfected or transfected with an FMRP-Flag plasmid. At 12 h.p.i. of WSN (MO1=1), cells were lysed and subjected to immunoprecipitation and western blot analyses. (c) FMRP interacts with NP in an RNA-dependent manner. 293T cells were transfected with TAP-NP or FMRP-Flag plasmids. Cell lysates were left untreated or were individually treated with RNase A for 1 h before being mixed together and TAP purified. (d) FMRP does not associate with the RNA-binding-deficient NP mutant. 293T cells were transfected with indicated plasmids and were analysed as described in (b). (e) FMRP transiently interacts with RNP. 293T cells were transfected with indicated plasmids. Cells were lysed and left untreated or treated with 0.5% PFA followed by TAP purification. (f) FMRP does not affect 4P association. 293T cells were transfected with PB1-TAP, PA, PB2 and NP plasmids along with increasing amounts of FMRP-Flag plasmids. Cells were then lysed followed by TAP purification (mean±s.d. of three independent experiments). (g) FMRP stimulates influenza virus RNP assembly. Cells were transfeced with indicated plasmids to reconstitute viral RNP and were analysed as described in (f) (mean±s.d. of three independent experiments, *P<0.05, **P<0.01, two-tailed Student’s t-test). (h) Knockdown of FMRP reduces RNP assembly. 293T cells were transfected with control or FMRP-specific siRNA along with RNP reconstitution plasmids. Cells were then analysed as described in (f). (i) FMRP overexpression promotes virus production. Vero cells were transfected with a vector plasmid or FMRP-Flag plasmid. Cells were then infected with WSN (MOI=0.001). At 48 h.p.i., viral supernatants were harvested and then subjected to plaque assays (mean±s.d. of three independent experiments, *P<0.05, two-tailed Student’s t-test).