Persistent Infections in L Cells with Temperature-Sensitive Mutants of Reovirus

Abstract

Serial passage of reovirus temperature-sensitive (ts) mutant C(447) produced by passage 9 (P9) a heavily defective population of virus from which the double-stranded RNA genomic segments L1, L3 and M1 were largely missing. Viral cores obtained from this P9 population were heterogeneous with respect to buoyant density in CsCl gradients, suggesting that particles were present with different combinations of deleted segments. Similar observations were made with the E(320) ts mutant of reovirus. By serial passage P15, 90% of the E(320) viral population was defective and the major missing genomic segments were L1 and L3. Persistent infections were readily established in monolayer cultures of [mouse fibroblast] L cells with P9 of C(447) virus and P15 of E(320) virus and in [African green monkey kidney] Vero cells with P9 of C(447) virus. Under similar conditions persistent infections could not be initiated with defective-free populations of C(447) or E(320) viruses. The greater the capacity of defective virus in the population to interfere with viral growth, the more readily persistent infection was initiated. During their maintenance persistently infected cells were subcultured about twice a wk. More than 80% of the cells continuously produced virus. By subculture 6 the original ts infectious viral component was replaced by a small-plaque mutant with a ts+ phenotype. Defective virus was always present in the carrier cells. Besides the more commonly observed defectives whose cores banded at about .rho. = 1.40 to 1.415 g/ml in CsCl gradients, a new class of defective core was seen banding in the region of 1.34 to 1.36 g/ml. This latter particle, which was not thoroughly characterized, is termed light defective. Persistently infected cells underwent periodic crises during their maintenance, during which the cultures partially lysed and then rapidly grew to confluence. Crises corresponded to a burst of infectious virus from the cells and a relatively low concentration of light defectives. During quiescent periods the concentration of light defectives amounted to as much as 98% of the total viral population. The function of light defectives is not yet clear, but it seems essential to assign major importance to defective virus in maintaining persistent infections in this system.