Electron microscopy of mammalian type-C RNA viruses: Use of conditional lethal mutants in studies of virion maturation and assembly

Mouse embryo cultures infected with each of a number of physiologically-distinct temperature-sensitive mutants of R-MuLV were examined by electron microscopy in an attempt to further define the replication defects of these mutants at the nonpermissive temperature (39°). Three mutants, ts24, ts25, and ts26, previously shown to be defective in postintegration steps, have been divided into two distinct subclasses. Mutants ts25 and ts26 synthesize viral antigens, but do not assemble viral particles at the nonpermissive temperature, whereas in mutant ts24 infected cells, particles at a late stage of budding were observed at very high frequency. An additional R-MuLV mutant, ts29, previously found to posses an early replication block associated with a thermolabile reverse transcriptase was shown to possess a second replication defect involving a very early stage of virus assembly. By examination of ts mutant infected cultures at various times following shift to their permissive temperatures partial synchronization was achieved with a high proportion of immature virions observed at similar stages of maturation. These findings more precisely define the stages of replication at which selected R-MuLV ts mutants are blocked at their nonpermissive temperatures, and illustrate the potential value of conditional lethal replication mutants for studies of type-C RNA virus maturation and assembly.