FIG 4.

Protein 4 is complexed with RNA. (A) Protein 4 migration in sucrose density gradients. RNA-protein complexes were extracted from TiLV-infected OmB cells, treated or not with RNase A, and separated in sucrose density gradients, by ultracentrifugation, according to the R-DeeP method. Fractions (1–23) of the sucrose gradients were collected from top to bottom (low to high density, respectively). The fractions were analyzed by Western blotting with αProtein4 antibodies. The black arrow at the bottom represents the shift of Protein 4 from dense to light fractions, following RNase A treatment. (B) Negative-staining immuno-EM of RNPs. Fractions of the gradient in panel A (–RNase, fractions 10 to 23), containing cellular RNPs that were extracted by the R-DeeP method and enriched for TiLV RNPs, were pooled, pelleted, and stained by immune-EM with primary αProtein4 antibodies and secondary gold particle-conjugated anti-rabbit antibodies (upper panel). Virion RNPs, extracted as described before (36), were stained as the cellular RNPs (lower panel).