Fig. 2.
Double-membrane layers induced by 1a/2apol contain 1a and 2apol and support BMV RNA replication and 1a-induced membrane association and nuclease resistance of viral RNAs. (A) Northern and Western blot analyses of RNA3, RNA4, 1a and 2apol accumulation in 1aG+2aA yeast that contain perinuclear spherules (Sph), or 1aG+2aG yeast that contain double-membrane layers (L). (B) ImmunoGold EM localization of 1a (Upper) and 2apol (Lower) in 1aG+2aG yeast containing double-membrane layers. Replication factor 1a was localized with polyclonal anti-1a antiserum (17). Because anti-2a antibodies gave weak ImmunoGold labeling, 2apol was localized by using an anti-GFP monoclonal antibody (JL-8, Clontech) to detect a 2apol-GFP fusion that supports BMV RNA replication (16) and induced ultrastructural changes indistinguishable from WT 2apol (compare with Fig. 1D). Nuc, nucleus; Cyto, cytoplasm. (Scale bars, 100 nm.) (C) Northern blot analysis of cell fractionation extracts from 1aG+2aA yeast that contain spherules (upper row) and 1aG+2aG yeast that contain double-membrane layers (lower row). The analysis shows the distribution of (+)RNA3 and (–)RNA3 in total lysate (T), 20,000 × g membrane-depleted supernatant (S), or membrane-enriched pellet (P) fractions after one of the following treatments: no additional treatment (none), addition of 0.01 units/ml micrococcal nuclease/1 mM CaCl2 for 15 min at 30°C (RNase), or addition of 0.5% Nonidet P-40 for 15 min at 4°C followed by nuclease treatment (Det/RNase).