Sensitive detection of morbillivirus cell-free transcription in a direct RNase protection assay

Cell-free measurement of viral transcription is necessary to determine if alterations of in situ levels of viral mRNA represent altered mRNA production or stability. Conditions for cell-free genomic transcription have been developed for the morbilliviruses canine distemper virus (CDV) and measles virus (MV), although the means for detecting nascent transcripts in these assays are insensitive in some cell systems. This work describes a technique in which CDV cell-free transcription reactions are modified so that non-radiolabeled transcripts are produced, precluding the need for limiting nucleotide concentrations in the reaction mixtures and allowing nucleotide concentrations which support optimal polymerase activities. Cell-free transcripts are then detected using anti-sense gene-specific riboprobes in an RNase protection assay (RPA). This approach is more sensitive than conventional slot blot analyses in detecting radiolabeled nascent transcripts and is efficacious in cell systems supporting inherently low levels of virus gene expression. Preformed viral RNA is distinguished from RNA synthesized during the course of the cell-free reactions by using a direct RPA (i.e., hybridization of target RNA prior to phenol/chloroform extraction). The use of this approach will expand the range of virus-host systems in which determinants of morbillivirus transcription can be characterized.