FIG. 3.

Analysis of protein synthesis and processing. (A) In vitro transcription and translation of nsP2 lethal (R615A, R699A, and K752A) and small-plaque mutants (K733A and R751A) using the rabbit reticulocyte lysate. Translation products were separated by SDS-PAGE and visualized by autoradiography. Locations of the proteins are indicated on the right. M indicates molecular mass (kDa). (B) Synthesis and processing of nsP2 in virus-infected cells. BHK cells in six-well plates were infected with wild-type (SINV) or mutant virus at an MOI of 5 and incubated at 30°C (I) or 37°C (II). At 12 h postinfection, cells were harvested and equal amounts of proteins were separated by SDS-10% PAGE. After transfer, the nitrocellulose membranes were processed by rabbit anti-nsP2 antibodies and an Alexa-Fluor 680-conjugated goat anti-rabbit secondary antibody (Molecular Probes). The K752A lane represents extracts obtained 12 h postelectroporation with Toto64 RNA carrying the mutation, which was tested previously along with mock and wild-type controls in a separate experiment.