Potato virus X‐based expression vectors are stabilized for long‐term production of proteins and larger inserts

Abstract

Plus-strand RNA viruses such as Potato virus X (PVX) are often used as high-yielding expression vectors in plants, because they tolerate extra transgene insertion and expression without disrupting normal virus functions. However, sequence redundancy due to promoter duplication often leads to genetic instability. Although heterologous subgenomic promoter-like sequences (SGPs) have been successfully used in Tobacco mosaic virus vectors, only homologous SGP duplications have been used in PVX vectors. We stabilized PVX-based vectors by combining heterologous SGPs from related potexviruses with an N-terminal coat protein (CP) deletion. We selected two SGPs with core sequences homologous to PVX, from Bamboo mosaic virus (BaMV) and Cassava common mosaic virus, as well as a SGP with a heterologous core sequence from Foxtail mosaic virus (FoMV). We found that only the BaMV and CsCMV SGPs were utilized by the PVX replicase. However, the transgene remained unstable, due to the presence of an additional region with strong sequence similarity at the 5' end of the cp gene. The BaMV SGP combined with an N-terminal CP deletion achieved high PVX vector stability. This new expression vector is particularly useful for long-term production of proteins and for larger inserts. The improved PVX-based vectors are suitable for the systemic expression of any gene of interest in PVX host plants. The PVX-based vector can be advantageous for the overexpression of proteins, to analyze protein functions in planta or as a system for virus-induced gene silencing.

A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize

“…In such PVX vectors, foreign genes or plant gene fragments for VIGS are inserted between ORF4 and ORF5 under the control of either a duplicated, native CP promoter or a heterologous CP promoter from a related Potexvirus spp. (Sablowski et al, 1995;Lacomme and Chapman, 2008;Dickmeis et al, 2014;Wang et al, 2014). Utilizing a similar strategy for CP promoter duplication in FoMV is complicated by the presence of ORF5A, which overlaps with ORF4.…”

“…The PVX vectors with duplicated promoters frequently suffer partial or complete loss of inserted sequences, especially when the insert size is large or the recombinant virus is passaged (Avesani et al, 2007;Dickmeis et al, 2014). In a PVX vector study involving heterologous subgenomic promoter-like sequences, a Bamboo mosaic virus subgenomic promoter combined with an N-terminal CP deletion resulted in the highest stability of foreign inserts following a passage to new plants (Dickmeis et al, 2014).…”

“…In a PVX vector study involving heterologous subgenomic promoter-like sequences, a Bamboo mosaic virus subgenomic promoter combined with an N-terminal CP deletion resulted in the highest stability of foreign inserts following a passage to new plants (Dickmeis et al, 2014). We tested the stability of inserts in the FoMV vector at the cloning site after the ORF5 stop codon, and no deletion was detected in L4 or L6 samples, but by L9 plants began to show evidence for deletions of the inserts.…”

“…The final ORF, ORF5, is expressed from sgRNA3 and encodes the only structural protein, the coat protein (CP), that is indispensable for virus assembly and cell-to-cell movement (Cruz et al, 1998). Importantly, viral vectors based on PVX have been developed for both gene expression and VIGS purposes, and they have facilitated functional genomics research in PVX host plants (Sablowski et al, 1995;Lacomme and Chapman, 2008;Dickmeis et al, 2014;Wang et al, 2014).…”

Design of virus-based nanomaterials for medicine, biotechnology, and energy

“…453 While larger inserts tend to result in genetic instability and loss of the gene of interest, 454 the combination of introducing a heterologous subgenomic promoter from bamboo mosaic virus and deleting a portion at the N-terminus of the CP was found to be a successful strategy to increase transgene expression stability. 455 …”