Genome Size Constraint in Replication and Packaging of Turnip Yellow Mosaic Virus

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. Previously, we have made the recombinant TYMV construct containing a 0.7 kb eGFP gene or a 1.8 kb GUS gene. The genomic RNAs from these constructs were efficiently encapsidated. To examine in more detail whether size constraint exists for replication and packaging of TYMV, we have inserted into the TY-GUS an extra sequence derived from either eGFP or GUS. We also made a recombinant containing RNA1 sequence of Flock house virus. These TYMV recombinants were introduced into Nicotiana benthamiana leaves by agroinfiltration. Northern blot analysis of the viral RNAs in the agroinfiltrated leaves showed that the genomic RNA band from the recombinant TYMV became weaker as longer sequence was inserted. The result also showed that the efficiency of genomic RNA encapsidation decreased sharply when an extra sequence of 2.2 kb or more was inserted. In contrast, the recombinant subgenomic RNA containing an extra sequence of up to 3.2 kb was efficiently encapsidated. Overall, these results show that size constraint exists for replication and encapsidation of TYMV RNA.

Characterization of a Replication Element in the Coat Protein ORF of Turnip Yellow Mosaic Virus

Turnip yellow mosaic virus (TYMV) is a non-enveloped icosahedral virus that has a single 6.3 kb positive-strand RNA as a genome. Previously, it was observed that the recombinant construct TY-eGFP2, where an eGFP gene was inserted at the position downstream of the coat protein (CP) ORF of TYMV genome, barely replicated. The inhibition of replication was relieved by insertion of an additional copy of the 3' quarter of the CP ORF after the foreign sequence. In this study, we have examined if the 3' quarter of the CP ORF contains any replication elements. M-fold analysis predicted three stem-loop structures in this region. Analysis of the TY-eGFP2 constructs containing one or two of these stem-loop structures indicates that the secondary structure predicted in the region between nt-6139 and nt-6181, termed SL2, is essential for TYMV replication. The critical role of SL2 was confirmed by the observation that deletion of the 3' quarter of the CP ORF from the wild-type TYMV genome nearly abolished replication and that insertion of SL2 into the deletion mutant restored the replication. Mutations disrupting the stem of SL2 greatly reduced viral RNA replication, indicating that the secondary structure is essential for the enhancing activity.