Spurious polyadenylation of Norovirus Narita 104 capsid protein mRNA in transgenic plants

Noroviruses are members of the family Caliciviridae, and cause a highly communicable gastroenteritis in humans. We explored the potential to develop a plant-based vaccine against Narita 104 virus, a Genogroup II Norovirus. In stably transgenic potato, we obtained very poor expression of Narita 104 virus capsid protein (NaVCP) despite the use of a strong constitutive promoter (dual enhancer 35S) driving the native coding sequence. We identified potentially detrimental sequence motifs that could mediate aberrant mRNA processing via spurious polyadenylation signals. Northern blots and RT-PCR analysis of total RNA revealed truncated transcripts that suggested premature polyadenylation. Site-directed mutagenesis to remove one potential polyadenylation near-upstream element resulted in an increased expression of NaVCP when transiently expressed in leaves of Nicotiana benthamiana. Further, cloning of the truncated cDNAs from transgenic NaVCP potato plants and transiently transfected N. benthamiana allowed us to identify at least ten different truncated transcripts resulting from premature polyadenylation of full length NaVCP transcripts. Comparative studies using real time PCR analysis from cDNA samples revealed lower accumulation of full length transcripts of NaVCP as compared to those from a gene encoding Norwalk Virus capsid protein (a related Genogroup I Norovirus) in transiently transfected plants. These findings provide evidence for impaired expression of NaVCP in transgenic plants mediated by spurious polyadenylation signals, and demonstrate the need to scrupulously search for potential polyadenylation signals in order to improve transgene expression in plants.