FIG. 5.

Processing of nsP4 by Ala183 and Arg183 (Toto1101) viruses. Cells were infected with Ala183 or Toto1101 at an MOI of 100 and were shifted to 40°C at 5 and 3.5 h p.i., respectively, when the maximum amount of nsP translation was occurring (data not shown). Cultures were pulse-labeled at 40°C immediately following shift with 100 mCi of [³⁵S]methionine/ml for 5 min and chased in the presence of a 20-fold excess of unlabeled methionine for 5, 15, 30, or 60 min before the samples were harvested. Cell extracts were immunoprecipitated with anti-nsP4 antibodies, and the immunoprecipitates were analyzed by polyacrylamide gel electrophoresis (Materials and Methods). The amount of cleaved nsP4 in each sample, quantified by scanning radioautographs of fluorographed gels, is expressed in relative units.