Noncytocidal persistent infections at 37 C of mouse L cells (LX X) with infective B particles of vesicular stomatitis virus (VSV) could be established only in the presence of large numbers of defective interfering (DI) particles. Under these conditions, there was a rapid spontaneous selection of temperature-sensitive (ts) virus. At 10 days there was an increase to 17.8% in the frequency of ts clones in the virus population; by 17 days this frequency had reached 85.2%, and by 63 days 100% of the clones isolated were ts at 39.5 C, the nonpermissive temperature used. All 34 of the clones isolated from the 84-day fluid had an RNA-phenotype, and 8 clones that were tested all belonged to VSV complementation group I. When tested by an interference assay, L,,fluids did not contain significant numbers of DI particles (<1 DI/PFU). Furthermore, persistent infection of L cells at 37 C could be initiated under conditions in which few, if any, DI particles were present by using low input multiplicities (10-4 and 10-5) of a clonal isolate of an RNA group I mutant obtained from LX ,\ cells. On the basis of these and other results, a mechanism is proposed to explain the role of ts mutants in both the establishment and maintenance of the persistently infected state. MATERIALS AND METHODS Cells. Primary chicken embryo (CE) cells, mouse L cells (clone 929), and a line (BHK-21) of hamster kidney cells were propagated in Eagle minimal essential medium plus 4% calf serum. Viruses. The large-plaque mutant of VSVINI) (L,VSV) described by Wertz and Youngner (22) was grown in BHK-21 cells and assayed in CE cells. Monolayers in 32-ounce (ca. 950-ml) culture bottles were infected with less than 0.01 PFU/cell to avoid production of DI particles. This wild-type virus stock is referred to in the text as VSVe. Analysis of [PH1uridine-labeled viral particles by sucrose gradients failed to detect DI particles in lysates produced under these conditions. ts mutants of VSVINI) 90

Temperature-sensitive (ts) mutants of vesicular stomatitis virus belonging to complementation groups I, II, and IV inhibited the replication of wild-type vesicular stomatitis virus when mixed infections were carried out in BHK-21 cells at 32, 37, and 39.5 C. The group IV mutant (ts G 41) was most effective in this regard; wild-type virus yields were inhibited almost 1,000-fold in mixed infections with this mutant at 32 C. In the case of group I and II mutants, inhibition of wild-type virus replication at 37 and 39.5 C was accompanied by an enhancement (up to 15,000-fold) of the yields of the coinfecting ts mutant. The yields of the group IV mutant (ts G 41) were not enhanced by mixed infections with wild-type virus at any temperature, although this mutant inhibited wildtype virus replication at all temperatures. The dominance of the replication of ts mutants at 37 C provides a rationale for the selection and maintenance of ts virus in persistently infected cells.

Evidence is presented which confirms that temperature-sensitive (ts) mutants with an RNA-phenotype are spontaneously selected in persistent infection of cell lines with Newcastle disease virus. Persistently infected BHK-21 cells, maintained since 1973, produce no interferon and are completely susceptible to vesicular stomatitis virus. Persistent infection of a canine kidney cell line (MDCK) terminated with destruction of all cells at about 100 days. Even under these conditions, a high proportion (33%) of RNA-temperature-sensitive mutants was present in the virus population 60 days after the infection was initiated.