Fig. 3

Nuclear RIG-I senses nuclear viral replication during RNP reconstitution. a 293T cells were RNP reconstituted (PR8 NA segment) in the presence of GFP, SLN-RIG-I, or NLS-RIG-I for 24 h and were subjected to cellular fractionation. The levels of viral RNA species in the cytoplasmic (Cyto) and nuclear (Nuc) fractions were determined by strand-specific qRT-PCR. Fold change is expressed using the ΔΔCt method relative to the nuclear RNA fraction of the PB1a reconstitution. b RNP reconstituted 293T cells (PR8 M segment) were subjected to immunofluorescence for FLAG-RIG-I (green) and FISH for M vRNA (red) at 24 h.p.t. Nuclei were stained with DAPI (blue). Scale bar = 10 μm. c 293T cells were cotransfected with plasmids encoding GFP or various RIG-I constructs along with p125Luc and pTK-rLuc followed by transfection with a 19-bp 5′ppp-dsRNA. RLUs are expressed as fold change relative to the GFP group without RNA stimulation (lane 1). Expression levels of FLAG-RIG-I/2CARD and GFP were determined by immunoblotting. Significant differences between mock-transfected and dsRNA-transfected cells expressing GFP or either RIG-I constructs were determined by two-way ANOVA followed by Sidak post-test. d, e 293T cells were RNP reconstituted with a Pol I vector or each of the eight viral segments in the presence of GFP, SLN-RIG-I, or NLS-RIG-I for 24 h. RLUs are expressed as fold change relative to the Pol I vector reconstitution with GFP. Significant differences between reconstitutions with viral segments and Pol I vector were determined by one-way ANOVA followed by Tukey post-test. f 293T cells were RNP reconstituted (PR8 NA segment) using either inactive (PB1a), WT, transcription-defective (T-), or replication-defective (R-) polymerases in the presence of GFP, SLN-RIG-I, NES-RIG-I, or NLS-RIG-I for 24 h. RLUs are expressed as fold change relative to the PB1a reconstitution in the presence of GFP. g The levels of viral RNA species in RNP reconstituted cells (f) were determined by strand-specific qRT-PCR and are expressed as percentage changes compared to the reconstitution using WT polymerase. Data are shown as mean ± SD of three experiments. ***p < 0.001; ****p < 0.0001; n.s. not significant