Fig. 5

RYSV N binds to importin α3 to enter the nucleus of the host cell. a Interactions of N and importin α1, importin α2, or importin α3, as determined using a yeast two-hybrid assay. Yeast cells co-transformed with pGBKT7–53 and pGADT7-T were used as a positive control. Yeast cells co-transformed with pGBKT7-Lam/pGADT7-T, pGBKT7-Importin α3/pGADT7-T were used as a negative control. b GST pull-down assay of the interaction of RYSV N with importin α3. Bait protein N was fused with GST-tagged protein. Prey protein importin α3 was fused with His-tagged protein. Purified GST incubated with prey protein and purified His incubated with bait protein were used as controls. c VCMs were transfected with dsP3 or importin α3 and inoculated with RYSV. At 72 h post RYSV inoculation, the cells were immunolabeled with N-specific IgGs directly conjugated to FITC. Scale bars: 25 μm. d RT-qPCR assay showing the relative transcript levels of the N, P, M, P3, and importin α3 genes during RYSV infection of N. cincticeps. N. cincticeps cells were transfected with dsImportin α3 or dsGFP and then inoculated with RYSV. Three biological repeats were performed. Data represent means ± SD and were analyzed using Student’s t-test; *P < 0.05; **P < 0.01. e Immunoblots of viral proteins from VCMs transfected with dsRNAs. N-, P-, M-, and P3-specific antibodies were used to detect N, P, M, and P3 protein, respectively, at 84 hpi. Actin was used as the control and was detected with actin-specific antibody