HIGH-LEVEL EXPRESSION OF EXOGENOUS GENES BY REPLICATION-COMPETENT RETROVIRUS VECTORS WITH AN INTERNAL RIBOSOMAL ENTRY SITE

We report the construction of two types of Rous sarcoma virus (RSV)-ba sed replication-competent avian retrovirus vectors, IR1 and IR2 to exp ress an exogenous gene at a very high level. In these vectors, the int ernal ribosomal entry site (IRES) derived from encephalomyocarditis vi rus (EMCV) was inserted between the env gene and an exogenous gene. Th e IR1 vector retains the splicing acceptor site that is present in the downstream of the env gene while the IR2 vector lacks it. Using a v-f os mutant (v-fos-CD3) as an example of exogenous genes, we show here t hat both IR1 and IR2 vectors expressed the gene product, CD3, at expre ssion levels 5- and 8-fold higher than that of their parental vector w ithout IRES, respectively. These vectors were moderately stable and ke pt a high-level expression of CD3 for at least three passages through the cells. Analysis of viral transcripts indicate that exogenous genes carried by both IR vectors were translated exclusively from the IRES that is present in all the species of the viral transcripts. High-leve l expression of exogenous genes was also observed in the case of the H oxa-13 gene in the IR1 vector or the fra-2 gene in the IR2 vector, ind icating that the extremely high-level expression characteristic of the se vectors is applicable to several exogenous genes. (C) 1997 Elsevier Science B.V.