Fig. 2

Growth deficiency of NP mutant virus rK229R. a Subcellular localization of NP in MDCK II cells infected with the indicated viruses at an MOI of 5 after 4, 6 and 8 h.p.i. Scale bar = 4 µm. b Cytoplasmic and nuclear accumulation of viral protein and RNA. MDCK II cells were infected at an MOI of 5 and 6 h.p.i., nuclear and cytoplasmic fractions were obtained. Upper panels show protein levels determined by Western blot analysis. β-Tubulin and histone H3 were used as markers for specific fractionation. Lower panels: relative vRNA transcript levels (PB2, HA, NP) in the nuclear and cytoplasmic fractions determined by quantitative RT–PCR. *p < 0.05. Error bars indicate the mean and s.d. of at least three independent experiments. Student’s t-test was used for two-group comparisons. c–e Fluorescence in situ hybridization to examine co-segregation of vRNAs in wt SC35M and rK229R-infected cells. MDCK II cells were infected at an MOI of 5 for 6 h and vRNAs were subsequently stained with segment-specific and fluorescently labeled probes. c Total cytoplasmic foci, positive for either one (HA, NP or PB2), two (HA and NP; HA and PB2; NP and PB2) or three vRNAs (HA, NP and PB2). d Composition of cytoplasmic foci positive for only one vRNA. e Cytoplasmic foci, positive for two co-segregating vRNAs. Each dot represents the total cytoplasm of a single cell. Depicted are 12–14 infected cells per virus from >4 independent infections. A two-tailed Mann–Whitney test was used for two-group comparisons