Analysis of spliced and unspliced rous sarcoma virus RNAs early and late after infection of chicken embryo fibroblasts: effect of cell culture conditions

Abstract

Quantitative determinations of spliced and unspliced viral RNA early after retrovirus infection or after transient transfection with proviral DNA have been difficult because of low intracellular viral RNA levels. In this report we describe conditions for the sensitive and quantitative assay of unspliced viral RNA and spliced mRNAs from avian sarcoma virus-infected and transiently transfected chicken embryo fibroblasts. RNase protection mapping was performed with a tandem 32P-labeled riboprobe consisting of three separate regions complementary to the major 5′ splice site and the two major 3′ splice sites ( env and src). The steady-state levels of viral RNA species were determined as early as 14 hr postinfection; no significant changes were observed in the relative levels of spliced mRNAs or in splice site usage as the infection progressed from 14 to 48 hr. After the cells became morphologically transformed at approximately 72 hr postinfection, the condition of the medium of the transformed cell culture affected the steady-state levels of viral RNAs. Unspliced genomic RNA predominated in the transformed cells at 4.5 to 8 hr after feeding; spliced env mRNA was the major species of RNA at 46 to 103 hr after medium change. In contrast, the steady-state levels of viral RNA in transiently transfected cells or in cells infected with a transformation-defective avian sarcoma virus were not as sensitive to the conditions of medium change. The medium-dependent changes in steady-state levels of viral RNA species may either be caused by a declining transcription rate resulting in the accumulation of the more stable env mRNA relative to the more labile unspliced RNA and src mRNA or to changes in the extent of viral RNA splicing.