CHARACTERIZATION OF AN INTERNAL RIBOSOMAL ENTRY SEGMENT WITHIN THE 5'-LEADER OF AVIAN RETICULOENDOTHELIOSIS VIRUS TYPE-A RNA AND DEVELOPMENT OF NOVEL MLV-REV-BASED RETROVIRAL VECTORS

The murine leukemia virus (MLV)-related type C viruses constitute a ma jor class of retroviruses that includes numerous endogenous and exogen ous mammalian viruses and the related avian spleen necrosis virus (SNV ). The MLV-related viruses possess a long and multifunctional 5' untra nslated leader involved in key steps of the viral life cycle-splicing, translation, RNA dimerization, encapsidation, and reverse transcripti on. Recent studies have shown that the 5' leader of Friend murine leuk emia virus and Moloney murine leukemia virus can direct cap independen t translation of gag precursor proteins (Berlioz et al., 1995; Vagner ed al., 1995b). These data, together with structural homology studies (Koning et al., 1992), prompted us to undertake a search for new inter nal ribosome entry segment (IRES) of retroviral origin. Here we descri be an IRES element within the 5' leader of avian reticuloendotheliosis virus type A (REV-A) genomic RNA. Data show that the REV-A 5' IRES el ement maps downstream of the packaging/dimerization (E/DLS) sequence ( Watanabe and Temin, 1982; Darlix et al., 1992) and the minimal IRES se quence appears to be within a 129 nt fragment (nucleotides 452-580) of the 5' leader, immediately upstream of the gag AUG codon. The REV-A I RES has been successfully utilized in the construction of novel high t iter MLV-based retroviral vectors, containing one or more IRES element s of retroviral origin. These retroviral constructs, which represent a starting point for the design of novel vectors suitable for gene ther apy, are also of interest as a model system of internal translation in itiation and its possible regulation during development, cancer, or vi rus infection.