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

Abstract

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.

Modification of Turnip yellow mosaic virus coat protein and its effect on virion assembly

“…Previously, we have observed that replication was nearly abolished when a foreign sequence was inserted between the CP ORF and the 3’-TLS (23) . The same result was obtained when the sequence between nt-6139 and nt-6181, which is proximal to the end of CP ORF, was deleted (24) , suggesting that a cis-acting element required for efficient replication exists near the end of CP ORF. In the TY-CP-M, the insertion was presumed to disturb some interactions involving the replication element.…”

Read-through Mutation in the Coat Protein ORF Suppresses Turnip Yellow Mosaic Virus Subgenomic RNA Accumulation

“…Deletion mutant analysis indicated that a sequence between nt-6139 and nt-6181, which was predicted to take a stemloop structure, was important in promoting TYMV replication (15). Subsequent examination of various mutant constructs of the potential replication element resulted in unexpected findings.…”

“…TY-eGFP2 and TY-eGFP2+CP 6203 constructs have been described previously (15). The construct TYΔCP2 was made by replacing the sequence between SmaI and EcoRI sites of the TY-eGFP2+CP 6203 with the linker ΔCP2, which was composed of two oligonucleotides: 5'-CCGGGTCACTAG-TACGCGTG-3' and 5'-AATTCACGCGTACTAGTGAC-3'.…”

“…Previous deletion mutant analysis showed that the sequence between nt-6139 and nt-6181, predicted to form a stem-loop with an asymmetric bulge, was critical to the replication of TYMV (15). The stem-loop structure was termed SL2.…”

“…When the sequence between nt-6067 and nt-6202 was deleted from wild-type TYMV (TYΔCP2), sgRNA and its product CP were hardly detected. Replication was recovered to 5~10% of wild type, when the sequence representing the SL2 was inserted into the TYΔCP2 (15).…”