Ambisense gene expression from recombinant rabies virus: random packaging of positive- and negative-strand ribonucleoprotein complexes into rabies virions

Abstract

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 chloramphenicol acetyltransferase (CAT) reporter gene from the antigenome. Transcription of the antigenome CAT gene was terminated by a modified RV gene junction able to mediate transcription stop and polyadenylation but not reinitiation of downstream transcripts. While in standard RV-infected cells genome and antigenome RNAs were present in a 49:1 ratio, the ambisense virus directed synthesis of equal amounts of genome and antigenome RNA (1:1). Total replicative synthesis was reduced by a factor of less than 2, revealing an unexpectedly high level of replication activity of the transcriptionally active promoter in the absence of the parental antigenome promoter. Successful packaging of ambisense ribonucleoprotein complexes (RNPs) into virions demonstrated that the parental 5 end of the RV genome RNA does not contain putative signals required for incorporation into virions. As determined both for standard RV and ambisense RV, virus particles contained genome and antigenome RNPs in the same ratios as those present in infected cells (49:1 and 1:1, respectively), indicating indiscriminate incorporation of RNPs independent of signals in the RNA. Ambisense expression of multiple foreign genes from RV vectors may circumvent problems with transcriptional attenuation of rhabdovirus housekeeping genes.

Roles of the respiratory syncytial virus trailer region: Effects of mutations on genome production and stress granule formation

“…Recombinant viruses that are analogous or similar to the LeC virus described here have also been generated for rabies and Sendai viruses (Finke and Conzelmann, 1997;Le Mercier et al, 2002). In contrast to the data described here, studies with these two viruses showed that the TrC promoter is only stronger than the Le promoter if the two promoters are in competition with each other.…”

“…This raised the possibility that there is discrimination between genome and antigenome-sense nucleocapsids at the step of virus assembly. However, studies with rabies and Sendai virus found that the Tr region does not contain a unique essential sequence for viral assembly, and indeed in the case of rabies virus, it was shown that the ratio between genomes and antigenomes in released viruses correlated with genome/antigenome ratios in the producer cells (Finke and Conzelmann, 1997;Le Mercier et al, 2002). The data obtained for RSV are in line with the results from rabies and Sendai viruses, as even though the LeC virus was impaired in growth, it could be rescued and propagated, demonstrating that it was capable of directing virus assembly and release.…”

Interplay between Innate Immunity and Negative-Strand RNA Viruses: towards a Rational Model

“…This dsRNA strongly activates the antiviral response, which leads to selection of SeV with a disabled antisense transcription promoter (147). Such ambisense SeV and RABV can be easily rescued and grow well only in IFN-disabled cells (76,147), indicating that there is strong selective pressure for avoiding the ambisense coding strategy by Mononegavirales due to RLRdependent innate immunity.…”

Overlapping Signals for Transcription and Replication at the 3′ Terminus of the Vesicular Stomatitis Virus Genome

“…The antigenomic-sense 9BN(ϩ) replication products (whose relative migration position in this gel is indicated by the arrow in Fig. 2A) were barely detectable even upon longer exposure of the fluorogram because the genomicsense replication products accumulate at much greater levels (Ͼ90%) than the antigenomic-sense replication products (14,30). From the data shown in Fig.…”