Differential transcription attenuation of rabies virus genes by intergenicregions: Generation of recombinant viruses overexpressing the polymerase gene

Gene expression of nonsegmented negative-sense RNA viruses involves sequent ial synthesis of monocistronic mRNAs and transcriptional attenuation at gen e borders resulting in a transcript gradient. To address the role of the he terogeneous rabies virus (RV) intergenic regions (IGRs) in transcription at tenuation, we constructed bicistronic model RNAs in which two reporter gene s are separated by the RV NIP gene border, Replacement of the 2-nucleotide (nt) NIP IGR with the 5-nt IGRs from the P/M or MIG border resulted in atte nuation of downstream gene transcription to 78 or 81%, respectively. A seve re attenuation to 11% was observed for the 24-nt G/L border. This indicated that attenuation in RV is correlated with the length of the IGR, and, in p articular, severe downregulation of the L (polymerase) gene by the 24 nt IG R, By reverse genetics, we recovered viable RVs in which the strongly atten uating G/L gene border of wild-type (wt) RV (SAD L16) was replaced with N/P -derived gene borders (SAD T and SAD T2), In these viruses, transcription o f L mRNA was enhanced by factors of 1.8 and 5.1, respectively, resulting in exaggerated general gene expression, faster growth, higher virus liters, a nd induction of cytopathic effects in cell culture. The major role of the I GR in attenuation was further confirmed by reintroduction of the wt 24-nt I GR into SAD T, resulting in a ninefold drop of L mRNA. The ability to modul ate RV gene expression by altering transcriptional attenuation is an advant age in the study of virus protein functions and in the development of gene delivery vectors.