The genomic retroviral RNA is the messenger for the translation of the gag and pol genes encoding the precursors to the major structural proteins and enzymes, respectively, of the virion core. The long 5 untranslated region, the leader, is formed of independent well-structured domains involved in key steps of the viral life cycle such as the initiation of proviral DNA synthesis, genomic RNA dimerization and packaging, and the initiation of gag translation. These functional features and the

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... nce of stable secondary structures between the cap and the gag initiation codon suggested that translation initiation of gag might proceed through a mechanism different from the canonical ribosome scanning process. Interestingly enough, murine leukemia viruses code also for a glycosylated gag precursor, named glyco-gag, initiated at a CUG codon upstream and in the same open reading frame as the AUG gag . We have investigated the translation initiation of gag and glyco-gag precursors of Friend murine leukemia virus (F-MLV) in the rabbit reticulocyte lysate system and in murine cells. Through site-directed mutagenesis of gag and glyco-gag initiation codons, we show that initiation of gag and glyco-gag synthesis does not utilize the classical ribosome scanning. When poliovirus protease 2A is coexpressed in murine cells, expression of MLV-lacZ RNA is not modified, indicating that translation initiation of MLV gag precursors is a cap-independent mechanism. In addition, the F-MLV leader was inserted between two genes in a dicistronic neo-MLV-lacZ mRNA, and its ability to promote expression was examined in vitro and in vivo. Results obtained demonstrate that an internal ribosome entry mechanism promotes translation of F-MLV gag precursors. This finding led us to construct a new dicistronic retroviral vector in which the F-MLV leader can promote both packaging of recombinant genomic RNA and expression of the 3 gene.