VIRUSES AND CELLS WITH MUTATIONS AFFECTING VIRAL ENTRY ARE SELECTED DURING PERSISTENT ROTAVIRUS INFECTIONS OF MA104 CELLS

To better understand mechanisms of persistent rotal virus infections o f cultured cells, we established independent, persistently infected cu ltures of MA104 cells, using rota virus strain SA11. The cultures were either passaged when the cells reached confluence or supplemented wit h fresh medium every 7 days, Viral titers in culture lysates varied fr om 10(4) to 10(7) PFU per ml during 350 days of culture maintenance. T rypan blue staining indicated that 72 to 100% of cells in the cultures were viable, and immunocytochemical staining using a monoclonal antib ody directed against viral protein VP6 demonstrated that 38 to 63% of the cells contained rotavirus antigen, We tested the capacity of rotav iruses isolated from the persistently infected cultures (PI viruses) t o infect cells cured of persistent infection, Although wild-type (wt) and PI viruses produced equivalent yields in parental MA104 cells, PI viruses produced greater yields than wt virus in cured cells, which in dicates that viruses and cells coevolve during persistent rotavirus in fections of MA104 cells. To determine whether mutations in viruses and cells selected during these persistent infections affect viral entry, we tested the effect of trypsin treatment of the viral inoculum on gr owth of wt and PI viruses. Trypsin pretreatment is required for postat tachment penetration of rotavirus virions into cells, In contrast to t he case with wt virus, PI viruses produced equivalent yields with and without trypsin pretreatment in parental MA104 cells, However, PI viru ses required trypsin pretreatment for efficient growth in cured cells. These results indicate that mutant viruses and cells are selected dur ing maintenance of persistent rotavirus infections of MA104 cells and suggest that mutations in each affect trypsin-dependent steps in rotav irus entry.