Virus promoters determine interference by defective RNAs: Selective amplification of mini-RNA vectors and rescue from cDNA by a 3 ' copy-back ambisense rabies virus

Typical defective interfering (DI) RNAs are more successful in the competit ion for viral polymerase than the parental (helper) virus, which is mostly due to an altered DI promoter composition. Rabies virus (RV) internal delet ion RNAs which possess the authentic RV terminal promoters, and which there fore are transcriptionally active and can be used as vectors for foreign ge ne expression, are poorly propagated in RV-infected cells and do not interf ere with RV replication. To allow DI-like amplification and high-level gene expression from such mini-RNA vectors, we have used an engineered 3' copy- back (ambisense) helper RV in which the strong replication promoter of the antigenome was replaced with the 50-fold-weaker genome promoter. In cells c oinfected with ambisense helper virus and mini-RNAs encoding chloramphenico l acetyltransferase (CAT) and luciferase, mini-RNAs were amplified to high levels. This was correlated with interference with helper virus replication , finally resulting in a clear predominance of mini-RNAs over helper virus. However, efficient successive passaging of mini-RNAs and high-level report er gene activity could be achieved without adding exogenous helper virus, r evealing a rather moderate degree of interference not precluding substantia l HV propagation. Compared to infections with recombinant RV vectors expres sing CAT, the availability of abundant mini-RNA templates led to increased levels of CAT mRNA such that CAT activities were augmented up to 250-fold, while virus gene transcription was kept to a minimum. We have also exploite d the finding that internal deletion model RNAs behave like DI RNAs and are selectively amplified in the presence of ambisense helper virus to demonst rate for the first time RV-supported rescue of cDNA after transfection of m ini-RNA cDNAs in ambisense RV-infected cells expressing T7 RNA polymerase.