FIG 3 .

Processing of RRV-T48 and RRV-T48_A534V nonstructural polyproteins and analysis of virus-induced translational shutoff. (a and b) RRV-T48 and RRV-T48_A534V nonstructural polyproteins and their processing products were obtained using the TNT SP6 rabbit reticulocyte system and plasmids containing the RRV-T48- or RRV-T48_A534V-infected cell DNAs. (a) The in vitro-translated nonstructural polyproteins and the products of their processing were denatured by boiling in 1% SDS. (b) Products of in vitro translation were immunoprecipitated (IP) using antibodies against nsP1, nsP2, and nsP3 of SFV. The asterisk indicates the position of the P34 polyprotein. (c) BHK-21 cells were infected at an MOI of 10 with RRV-T48 or RRV-T48_A534V. At 4, 6, 8, 10, 12, 16, 20, and 24 h p.i., cells were labeled with medium containing 50 µCi [³⁵S]Met and [³⁵S]Cys. After labeling, cells were lysed in SDS loading buffer. In panels a to c, labeled proteins were separated by SDS-PAGE and visualized/quantified with a Typhoon imager. Data from one of two reproducible independent experiments are shown in each of the panels.