Late stage synchronization of respiratory syncytial virus replication

Abstract

Although a deficiency of phenylalanine (PA) in the medium prevented the production of infectious virus by Hela cells infected with respiratory syncytial (RS) virus, many of the early steps in virus replication were completed, and the infection was synchronized at a late stage. In the deficient medium (1 μg of PA per milliliter), protein was synthesized at the normal rate for about 6 hr, not long enough even for completion of the RS-virus latent period (10–12 hr). However, when infected cells were incubated in the PA-deficient medium for 16–18 hr the infection progressed to the stage of perinuclear inclusions containing viral RNA and protein. Experiments utilizing 2-thiouridine (which stops infectious RNA synthesis in HeLa cells infected with rhinovirus) demonstrated that all the viral RNA required for a full yield of virus was synthesized during the period of incubation in the deficient medium. When PA (5 μg/ml) was added to infected cells that had been incubated in the deficient medium for 18 hr, virus production started within 2 hr. The perinuclear inclusions enlarged, viral antigen appeared at the cell periphery within 1 hr, and within 3 hr the cell membrane contained viral antigen, and syncytia had begun to form. The data suggested that PA deficiency did not prevent the synthesis of viral nucleocapsid, but may have prevented the synthesis of viral envelope antigen, and further suggested that continuing protein synthesis was required for movement of the nucleocapsid to the cell periphery, as well as for the synthesis of viral envelope protein. The 2-hr interval between PA supplementation and the appearance of infective virus evidently represented the time required to incorporate the viral RNA into mature virions. PA is not a specific requirement for RS-virus production, as arginine deficiency also synchronized infection in HeLa cells.