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 transcripti on have been developed for the morbilliviruses canine distemper virus (CDV) and measles virus (MV), although the means for detecting nascent transcripts in these assays ace insensitive in some cell systems. Thi s work describes a technique in which CDV cell-free transcription reac tions are modified so that non-radiolabeled transcripts are produced, precluding the need for limiting nucleotide concentrations in the reac tion mixtures and allowing nucleotide concentrations which support opt imal polymerase activities. Cell-free transcripts are then detected us ing anti-sense gene-specific riboprobes in an RNase protection assay ( RPA). This approach is more sensitive than conventional slot blot anal yses in detecting radiolabeled nascent transcripts and is efficacious in cell systems supporting inherently low levels of virus gene express ion. Preformed viral RNA is distinguished from RNA synthesized during the course of the cell-free reactions by using a direct RPA (i.e., hyb ridization of target RNA prior to phenol/chloroform extraction). The u se of this approach will expand the range of virus-host systems in whi ch determinants of morbiliivirus transcription can be characterized.