Semliki forest virus mutants with temperature-sensitive transport defect of envelope proteins

Abstract

Seven RNA-positive temperature-sensitive mutants of Semliki Forest virus were studied to find mutants with a defect in the transport of envelope glycoproteins to the host cell plasma membrane. Surface immunofluorescence of infected cells was recorded at the restrictive temperature (39°) and after shift to the permissive temperature (28°) carried out in the presence or absence of protein synthesis inhibitors. Preformed envelope proteins of two mutants, ts-1 and ts-7, were found at the plasma membrane only after shift to the permissive temperature. Staining of intracellular antigens after mild detergent treatment of ts-1 and ts-7 infected cells showed that at 39° the envelope proteins had reached th Golgi apparatus. Electron microscopy of ts-1 and ts-7 infected cells, maintained at 39°, showed that all the nucleocapsids were free in the cytoplasm. In ts-7, but not in ts-1, infected cells binding of nucleocapsids and virus budding started soon after the cultures had been shifted to the permissive temperature. First the budding was observed only at the Golgi and later also at the plasma membrane. Envelope proteins labeled at the restrictive temperature were efficiently incorporated into ts-7 but not into ts-1 virus particles, released after shift to 28°. We suggest that the viral glycoproteins have different “signals” for the transport and for the binding of nucleocapsid. In ts-7 both “signals” become functional after shift to 28°, whereas in ts-1 only the “transport signal” defect is reversible.