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 major class of retroviruses that includes numerous endogenous and exogenous mammalian viruses and the related avian spleen necrosis virus (SNV). The MLV-related viruses possess a long and multifunctional 5′ untranslated leader involved in key steps of the viral life cycle—splicing, translation, RNA dimerization, encapsidation, and reverse transcription. Recent studies have shown that the 5′ leader of Friend murine leukemia virus and Moloney murine leukemia virus can direct cap independent translation of gag precursor proteins (Berlioz et al., 1995; Vagner et al., 1995b). These data, together with structural homology studies (Koning et al., 1992), prompted us to undertake a search for new internal ribosome entry segment (IRES) of retroviral origin. Here we describe 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 element maps downstream of the packaging/dimerization (E/DLS) sequence (Watanabe and Temin, 1982; Darlix et al., 1992) and the minimal IRES sequence 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 IRES has been successfully utilized in the construction of novel high titer MLV-based retroviral vectors, containing one or more IRES elements of retroviral origin. These retroviral constructs, which represent a starting point for the design of novel vectors suitable for gene therapy, are also of interest as a model system of internal translation initiation and its possible regulation during development, cancer, or virus infection.

Overview summary

Many strategies being developed for gene transfer with potential therapeutic applications require the co-expression of heterologous gene products. In this respect, internal ribosome entry segments (IRES) were found to be an efficient means of expressing two exogenous genes in cells without the need for two promoters or a regulated splicing mechanism. Internal translation initiation has been shown to direct synthesis of gag precursor proteins of Friend murine leukemia virus and Moloney murine leukemia virus (Berlioz et al., 1995; Vagner et al., 1995b) and IRES have been found in retrotransposons such as the rat VL30 region of the Harvey murine sarcoma virus leader (Berlioz et al., 1995). In this study, we describe the characterization of an IRES within the 5′ untranslated leader of avian reticuloendotheliosis virus type A RNA. In addition, we report the development of novel murine leukemia virus-based retroviral vectors, containing one or more IRESes of retroviral origin.