A FUNCTIONAL-ROLE FOR THE CONSERVED PROTONATABLE HAIRPINS IN THE 5'-UNTRANSLATED REGION OF TURNIP YELLOW MOSAIC-VIRUS RNA

The 5' untranslated region (UTR) of the RNA of several tymoviruses con tains conserved hairpins with protonatable internal loops, consisting of C-C and C-A mismatches (K. Hellendoorn, P. J. A. Michiels, R. Buite nhuis, and C. W. A. Pleij, Nucleic Acids Res. 24, 4910-4917, 1996). He re, we present a functional analysis of the 5' UTR of turnip yellow mo saic virus (TYMV) RNA, which contains two protonatable hairpins with n early identical internal loops, Mutations were introduced in an infect ious cDNA clone, and T7 RNA transcripts were used to infect Chinese ca bbage plants, Different symptoms were observed for the various mutants , pointing to a functional role of the C-C and C-A mismatches in the h airpins of the 5' UTR. The replication of the virus is influenced by t he mutations made, while in vitro translation studies showed that the expression of the two overlapping reading frames of TYMV is not influe nced by the secondary structure of the leader. Various mutants were pr opagated for up to five serial passages of infection, and the sequence of the 5' UTR was determined. This resulted in virus RNA with new non -wild-type sequences that produced the wild-type phenotype in infected plants, Remarkably, in all cases C-C or C-A mismatches were introduce d. The internal loop of the 5'-proximal hairpin seems to be more impor tant for the viral life cycle than that of the second hairpin, A delet ion of 75% of the leader, including the two hairpins, resulted in a vi rus that was deficient in viral spread. Since the ratio between filled and empty capsids was changed drastically by this mutation, a role of the 5' UTR in viral packaging is proposed.