SINDBIS VIRUS EXPRESSION VECTORS - PACKAGING OF RNA REPLICONS BY USING DEFECTIVE HELPER RNAS

Since the recovery of infectious RNA transcripts from full-length cDNA clones, alphavirus genome RNAs have been engineered to allow expressi on of heterologous RNAs and proteins. The highest levels of expression of heterologous products are achieved when the viral structural genes are replaced by the heterologous coding sequences. Such recombinant R NAs are self-replicating (replicons) and can be introduced into cells as naked RNA, but they require trans complementation to be packaged an d released from cells as infectious virion particles. In this report, we describe a series of defective Sindbis virus helper RNAs which can be used for packaging Sindbis virus RNA replicons. The defective helpe r RNAs contain the cis-acting sequences required for replication as we ll as the subgenomic RNA promoter which drives expression of the struc tural protein genes. In cells cotransfected with both the replicon and defective helper RNAs, viral nonstructural proteins translated from t he replicon RNA allow replication and transcription of the defective h elper RNA to produce the virion structural proteins. A series of defec tive helper RNAs were compared for the ability to package the replicon RNA as well as for the ability to be replicated and packaged. One def ective helper RNA not only packaged the replicon but also was itself e ncapsidated and would be useful under conditions in which extensive am plification is advantageous. Other defective helper RNAs were able to package the replicon efficiently but were packaged very poorly themsel ves. These helpers should be useful for applications in which expressi on of the viral structural proteins or virus spread is not desired.