Development and optimization of a Pepino mosaic virus-based vector for rapid expression of heterologous proteins in plants

Authors: Abrahamian, Peter; Hammond, John; Hammond, Rosemarie

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/11/2020
Publication Date: 1/4/2021
Citation: Abrahamian, P., Hammond, J., Hammond, R. 2021. Development and optimization of a Pepino mosaic virus-based vector for rapid expression of heterologous proteins in plants. Applied Microbiology and Biotechnology. 105:627-645. https://doi.org/10.1007/s00253-020-11066-0.
DOI: https://doi.org/10.1007/s00253-020-11066-0

Interpretive Summary: Plant virus-derived vectors are excellent transient expression systems for producing foreign proteins in plants, and versatile tools with many applications in the agricultural and medical fields. In this study, we developed pepino mosaic virus (PepMV) into an expression vector for protein production. Vector development was optimized by confirming effective replication and movement of virus within plants. Different expression strategies were explored for improving expression of the foreign gene of interest. The PepMV-based vector was compared to other closely related potexvirus-based vectors. The advantages of the PepMV-based vector are its rapid infection and high levels of protein production, and its stability in successive passages onto plants, which is helpful in large scale protein production. PepMV is mechanically transmitted and poses minimal biosafety risk with use of proper containment measures. The development of this vector is of interest to many audiences such as researchers in virus- and plant-based expression systems, biotechnology stakeholders and governmental organizations. This vector is an additional tool for exploiting plants as a safe and rapid manufacturing system for a multitude of proteins, such as antimicrobials and pharmaceuticals.

Technical Abstract: Plant-virus derived vectors are versatile tools with multiple applications in agricultural and medical biotechnology. In this study, we developed pepino mosaic virus (PepMV) (family Alphaflexiviridae; genus Potexvirus) into a vector for heterologous protein expression in plants. PepMV was initially cloned in a step-wise manner, fully sequenced and the full-length infectious clone was tested for infectivity in Nicotiana benthamiana. Initial infectious clones resulted in poor replication of PepMV and lack of systemic movement. Mutations in the viral sequence affected systemic infection. Two suspected mutations were altered for restoring systemic infectivity. PepMV infection was apparent as early as 4 days post inoculation (dpi) in N. benthamiana. A multiple cloning site was inserted into the PepMV genome for introduction and expression of foreign genes. Several modifications to the wild-type vector were made, such as a replacing the native subgenomic promoter (SGP) with a heterologous SGP, and introduction of translational enhancers and 3’UTR terminators, to improve heterologous expression of the foreign gene-of-interest. GFP was used as a reporter for monitoring virus infection and protein production. Strong GFP expression was observed as early as 4 dpi with a translational enhancer. The PepMV-based vector produces rapid expression of the foreign gene in comparison to two other potexvirus-based vectors. GFP production was monitored over time and optimal protein production was recorded at 7 dpi. GFP protein levels reached up to 3% and decreased to 0.5% total soluble protein at 7 and 14 dpi, respectively. Future studies will evaluate this virus-based vector for large scale production of pharmaceutical compounds.