The 5′-UTR of Turnip yellow mosaic virus does not include a critical encapsidation signal

Abstract

Turnip yellow mosaic virus (TYMV) RNA has two hairpins in the 5′ untranslated region (UTR) with internal C C and C A mismatches that become protonated and are able to base pair at a pH near 5. The protonatable hairpins have previously been implicated as playing an important role in RNA encapsidation. We have examined the role of the 5′-UTR in the amplification and packaging of TYMV RNA using agroinfiltration of Chinese cabbage leaves to express various TYMV constructs with mutations affecting the 5′-UTR and the two hairpins. Mutations affecting the protonatable centers of the two hairpins, as well as deletion of one or both hairpins and deletion or mutation of the 17-nucleotide region upstream of the hairpins decreased viral amplification to varying extents (c. 10- to 1000-fold). However, in all these cases, the viral RNAs present in non-denaturing leaf extracts were predominantly ribonuclease resistant, indicative of encapsidation. These results show that, while the 5′ hairpins are necessary for efficient amplification of TYMV, there appears to be no essential role for the 5′-UTR or its protonatable hairpins in the packaging of TYMV RNA. In a second set of experiments, it was demonstrated that TYMV can efficiently amplify in plants held in the dark, and that the progeny RNAs are efficiently encapsidated. Together, these observations argue for a revision of the model for TYMV encapsidation in which packaging occurs in low pH conditions that are generated by proton gradients produced by photosynthetic activity in the light and RNA packaging is dependent on the protonatable 5′ hairpins.