Mutational analysis of the predicted secondary RNA structure of the Mason-Pfizer monkey virus packaging signal

The 5′ end of the Mason-Pfizer monkey virus (MPMV) genomic RNA has been predicted to fold into a complex stem/loop structure that is thought to play a role in specific RNA encapsidation. In this study, we used a set of mutations that either abrogated or recreated the first four stem loops predicted within the 5′ untranslated region (5′ UTR) for effects on RNA packaging. Test of these mutations in our biological assay revealed that only stem loop 1 (SL1) was important for the packaging potential of MPMV, while mutations in none of the other stem loops affected packaging significantly. Interestingly, it was the primary sequence of SL1 RNA and not its secondary structure that affected packaging since compensatory mutations that reformed SL1 were unable to restore the packaging efficiency of the retroviral vector. Additionally, our mutational analysis reveals that stem loop 4, predicted to be the major packaging determinant of MPMV, does not seem to have a significant role in packaging. Finally, results of the biological effects of the structural mutations are discussed in relation to their effects on the folding potential of the various stem loops.