SINDBIS VIRUS REPLICONS AND SINDBIS VIRUS - ASSEMBLY OF CHIMERAS AND OF PARTICLES DEFICIENT IN VIRUS-RNA

Alphaviruses are a well-characterized group of positive-strand RNA vir uses. The identification of cis-acting elements in their genomes and t heir replication strategy have made them useful as vectors for the exp ression of heterologous genes. In infected cells, the nonstructural pr oteins, required for replication and transcription of the viral genes, are translated from the genomic RNA; the structural proteins, the cap sid protein that interacts with the RNA to form the nucleocapsid and t he proteins embedded in the lipid envelope, are translated from a subg enomic mRNA and can be replaced by heterologous genes. Such modified g enomes are self replicating (replicons); they can be introduced into t he cells by transfection and can also be packaged into extracellular p articles with defective helper (DH) RNAs. The particular DH RNA determ ines how well it is replicated and to what extent it is packaged. One potential complication of this system has been that recombination betw een the replicon genome and the DH RNA may occur. The studies describe d here were designed to prevent recombination by expressing the capsid protein from one DH RNA and the virus membrane proteins from a second helper RNA. Recombination to yield a nonsegmented infectious virus ge nome would then require several independent crossover events. There is a translational enhancer located downstream of the initiating AUG in the RNA of the capsid gene that had to be conserved in the second help er to achieve high-level expression of the viral glycoproteins. For th is reason, we modified the capsid protein gene in two ways: the first was to use the capsid protein gene from a different alphavirus, Ross R iver virus, and the second was to make deletions in that gene to maint ain the translational enhancer in the RNA but to eliminate the positiv ely charged region in the protein that should be essential for the spe cific and nonspecific interactions with RNA. Transfections with replic on RNA and the deleted chimeric DH RNA as the only helper resulted in the high-level production of particles that were almost completely dev oid of RNA. The inclusion of a helper expressing an intact Sindbis vir us capsid protein gene led to the production of high levels of package d replicons. Recombinants were not detected even after several undilut ed passages.