Fig. 4

Effect of DI RNA on the accumulation and encapsidation of viral RNAs. 293T cells were transfected with increasing amounts of the 1/244 DI PolI plasmid (0, 0.1, 0.5 and 1.0 μg) and a constant amount of the plasmids needed for the expression of infectious A/WSN virus. After co-cultivation with MDCK cells RNA was extracted from cells and from virus particles purified from culture fluids. a. RNA extracted from cell lysates (top panel) and virus particles from supernatants (lower panel) at 1, 2 and 3 days post co-cultivation was analysed with probes specific for segment 1 RNA, segment 7 RNA, and 1/244 DI RNA. The sizes of RNA markers are shown on the left and the identity of the RNAs on the right. b. Cell lysate RNA and virion RNA extracted on day 3 were analysed with probes specific for segment 2 RNA and segment 7 RNA. c. A/WSN infectivity in cell supernatants measured by microplaque assay. The infectivities on 1 (■), 2 (▲) and 3 (●) days after co-cultivation are shown. Data are the mean of 2 independent experiments with the bar representing the range. d. The ratios of levels of segment 1 RNA to segment 7 in virions on days 2 (■) and 3 (●) and of segment 2 RNA to segment 7 in virions on day 3 (▼) were determined by densitometry. These values were compared with the equivalent ratios determined from transfected cells. The ratios of segment 1 or segment 2 in virions over total cellular RNA were calculated and normalised to the value determined in the absence of 1/244 DI RNA