Recovery of infectious proviral DNA from mammalian cells infected with respiratory syncytial virus

Abstract

The DNA fraction from a line of bovine embryonic kidney cells originally exposed as primary cultures several months earlier to a temperature-sensitive (ts) mutant of respiratory syncytial (RS) virus could be used to transfect human HEp-2 cells with the production of infectious RS virus. The DNA donor cells, designated BEK/RS ts, retained their healthy fibroblastic appearance during continuous cultivation at a temperature (39 degrees) restrictive for growth of the original infecting mutant and showed no evidence for RS virus replication or viral antigen synthesis when directly examined for these activities by conventional methods. The infectious property of the DNA from BEK/RS ts cells was abolished by exposure of the nucleic acid preparation to DNase (but not RNase) or by pretreatment of recipient HEp-2 cells with actinomycin D or mitomycin C. The latter drug treatments substantially enhanced the replication of infecting wild-type RS virus in HEp-2 cells. Viral isolates derived from the progeny of a DNA transfection included clones possessing several genetic markers of the RS ts mutant originally used to infect BEK/RS ts cells and other virus clones that appeared to be either hybrid or wild-type for phenotypic properties such as their temperature sensitivity. An infectious proviral DNA was also detected in a line of virogenic HEp-2 cells (HEp-2/RS) persistently infected with respiratory syncytial virus after exposure to the wild-type strain 2 years earlier.