Complementation of defective reovirus by ts mutants

Abstract

Defective reovirions lacking the largest (L-1) of the normal 10 genomic segments grow only in association with helper reovirus. Because of the similarity in properties of defective and infectious virions, separation of the two populations by physical methods has been unseccessful. Controlled digestion of purified virus removes the outer capsomeres of the virions. The resulting core particles containing the viral genome have a buoyant density of 1.43/ml if derived from infectious virions and of 1.415g/ml if they originate in defectives, and this difference permits ready separation of the two types of cores. With the purpose of obtaining a pure population of defective virions, L cells were co-infected with defective cores and a class E temperature-sensitive mutant which has a mutation in an early function. After three serial passages at the permissive temperature (31 C) to build up the defective population, a fourth passage was made at 39 C, the nonpermissive temperature. The virus purified from this passage was predominantly defective; it contained practically no E mutant and had a low background of wild-type virus. Complementation was thus asymmetric; the L-1 function required for growth of defective virus was supplied by the E mutant and is thus a trans-function, while defective virus did not complement the E mutation which is thus in a cis-acting function. Defective virions were indistinguishable from infectious virions except for the absence of the L-1 genomic segment in the defectives. Such defective virions could be complemented at 39 C by class A and B temperature-sensitive mutants, both of which have lesions in late functions.