FIG. 3.

Altered migration of nsP3 proteins made at 40°C indicated that certain mutant nsP3 proteins were temperature sensitive for phosphorylation. (A) CEF cells were infected with either an nsP3 mutant, the revertant of ts4, or parental SIN HR and were maintained at 30°C or shifted to 40°C late in infection. Viral proteins were pulse-labeled with [³⁵S]methionine for 30 min as described above (Materials and Methods) followed by a chase period of 1 h before the cells were lysed. Infected-cell lysates were immunoprecipitated with nsP3 antibodies, and the precipitates were analyzed by gel electrophoresis. (B) Lysates from ts4-, Toto:ts7B5-, or mock-infected cells labeled and chased at 40°C were immunoprecipitated with antibodies specific for nsP3 (left panel) or for nsP4 (right panel), and the immunoprecipitates were analyzed on 5 to 10% Laemmli linear gradient polyacrylamide gels (31).