Abstract
P3N-PIPO (P3 N-terminal fused with Pretty Interesting Potyviridae ORF), the movement protein of potyviruses, is expressed as a translational fusion with the N-terminus of P3 in potyviruses. As reported in previous studies, P3N-PIPO is expressed via transcriptional slippage at a conserved G2A6 slippery site in the genus Potyvirus. However, it is still unknown whether a similar expression mechanism of P3N-PIPO is used in the other genera of the family Potyviridae. Moreover, due to the extremely low expression level of P3N-PIPO in natural virus-infected plants, the peptides spanning the slippery site which provide direct evidence of the slippage at the protein level, have not been identified yet. In this study, a potato virus X (PVX)-based expression vector was utilized to investigate the expression mechanism of P3N-PIPO. A high expression level of the P3N-PIPO(WT) of turnip mosaic virus (TuMV, genus Potyvirus) was observed based on the PVX expression vector. For the first time, we successfully identified the peptides of P3N-PIPO spanning the slippery site by mass spectrometry. Likewise, the P3N-PIPO(WT) of wheat yellow mosaic virus (WYMV, genus Bymovirus) was also successfully expressed using the PVX expression vector. Integrated proteome and transcriptome analyses revealed that WYMV P3N-PIPO was expressed at the conserved G2A6 site through transcriptional slippage. Moreover, as revealed by mutagenesis analysis, Hexa-adenosine of the G2A6 site was important for the frameshift expression of P3N-PIPO in WYMV. According to our results, the PVX-based expression vector might be used as an excellent tool to study the expression mechanism of P3N-PIPO in Potyviridae. To the best of our knowledge, this is the first experimental evidence dissecting the expression mechanism of a bymovirus P3N-PIPO in the experimental host Nicotiana benthamiana.
Highlights
Most RNA viruses have a relatively small genome size, which seems to be insufficient for RNA viruses to cope with long-term selective pressure
The behavior of P3N-PIPO M2 was similar to wild-type P3N-PIPO (WT). These results indicated that Hexa-adenosine was important for the maintenance of P3N-PIPO frameshift expression
Studies on P3N-PIPO frameshift expression have been limited in the genus Potyvirus, and it remains unknown whether a similar frameshift expression of P3N-PIPO occurs in other genera in Potyviridae
Summary
Most RNA viruses have a relatively small genome size, which seems to be insufficient for RNA viruses to cope with long-term selective pressure. Transcriptional slippage often takes place on repetitive nucleotides such as poly(A) or poly(T) tracts, resulting in the synthesis of heterogeneous mRNAs that always insert one or more extra nucleotides but seldom delete one or two bases [3]. If this occurs in a coding sequence, the heterogeneous mRNA will translate into more than one protein product. Owing to the extremely low expression level of P3N-PIPO in natural virus-infected plants, a reliable experimental system is needed to isolate sufficient frameshift-derived protein for analysis. Our research sheds more light on the expression mechanism of P3N-PIPO in the family Potyviridae, and provides an excellent tool to study the slippage efficiency of P3N-PIPO
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