Genetic and physiological characterization of a temperature-sensitive mutant of cowpea mosaic virus

A temperature-sensitive mutant of Cowpea mosaic virus (CPMV), N168, was isolated after nitrous acid treatment of CPMV-RNA. The mutant could be distinguished from wild-type CPMV by its symptoms on Cowpea and Pinto beans at 22° and practically complete lack of multiplication in cowpeas at 30°. Supplementation tests demonstrated that symptom changes and temperature sensitivity were due to one or more mutations in the RNA of the middle component of N168. The time course of appearance of virus-specific membrane-bound replicase in Cowpea plants inoculated with wild-type or mutant virus and grown at the permissive or nonpermissive temperatures demonstrated the absence of replicase activity at the restrictive temperature in plants infected with N168. From results on the temperature dependency and the thermostability of the wild-type and mutant membrane-bound replicase in vitro and the behavior of mutant replicase activity in plants upon a shift from the permissive to the nonpermissive temperature, it was concluded that the failure of mutant N168 to multiply at 30° was due to a defect in the synthesis or assembly of the virus RNA replicase, and not to temperature sensitivity of the replicase itself.