AMBISENSE GENE-EXPRESSION FROM RECOMBINANT RABIES VIRUS - RANDOM PACKAGING OF POSITIVE-STRAND AND NEGATIVE-STRAND RIBONUCLEOPROTEIN COMPLEXES INTO RABIES VIRIONS

We have recovered from cDNA a rabies virus (RV) containing identical, transcriptionally active promoters at its genome (negative-strand) and antigenome RNA and directing efficient expression of a chloramphenico l acetyltransferase (CAT) reporter gene from the antigenome. Transcrip tion of the antigenome CAT gene was terminated by a modified RV gene j unction able to mediate transcription stop and polyadenylation but not reinitiation of downstream transcripts. While in standard RV-infected cells genome and antigenome RNAs mere present in a 49:1 ratio, the am bisense virus directed synthesis of equal amounts of genome and antige nome RNA (1:1). Total replicative synthesis was reduced by a factor of less than 2, revealing an unexpectedly high level of replication acti vity of the transcriptionally active promoter in the absence of the pa rental antigenome promoter. Successful packaging of ambisense ribonucl eoprotein complexes (RNPs) into virions demonstrated that the parental 5' end of the RV genome RNA does not contain putative signals require d for incorporation into virions. As determined both for standard RV a nd ambisense RV, virus particles contained genome and antigenome RNPs in the same ratios as those present in infected cells (49:1 and 1:1, r espectively), indicating indiscriminate incorporation of RNPs independ ent of signals in the RNA. Ambisense expression of multiple foreign ge nes from RV vectors may circumvent problems with transcriptional atten uation of rhabdovirus housekeeping genes.