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

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 to restore systemic infectivity. PepMV infection was apparent as early as 4 days post agroinfiltration (dpa) inoculation 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 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 dpa 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 between 5 and 7 dpa. GFP protein levels reached up to 4% and decreased to 0.5% total soluble protein at 7 and 14 dpa, respectively. Future studies will evaluate this virus-based vector for large-scale production of pharmaceutical compounds. KEY POINTS: • A pepino mosaic virus isolate was developed into a plant-based expression vector. • Expression levels of the heterologous protein were comparable or exceeded previously developed viral vectors. • Protein levels in plants were highest between 5 and 7 days and decreased gradually.