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

Lolita G. Mathew, Bryan Maloney, Naokazu Takeda, Hugh Mason

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)263-275
Number of pages13
JournalPlant Molecular Biology
Volume75
Issue number3
DOIs
StatePublished - 2011

Fingerprint

Norovirus
Polyadenylation
Genetically Modified Plants
Capsid Proteins
coat proteins
transgenic plants
Viruses
viruses
Messenger RNA
Nicotiana benthamiana
Solanum tuberosum
Caliciviridae
Norwalk virus
Complementary DNA
Genotype
genetically modified organisms
potatoes
site-directed mutagenesis
gastroenteritis
Gastroenteritis

Keywords

  • Norovirus
  • Plant based vaccine
  • Plant viral vectors
  • Polyadenylation
  • Transgenic plants

ASJC Scopus subject areas

  • Plant Science
  • Agronomy and Crop Science
  • Genetics

Cite this

Spurious polyadenylation of Norovirus Narita 104 capsid protein mRNA in transgenic plants. / Mathew, Lolita G.; Maloney, Bryan; Takeda, Naokazu; Mason, Hugh.

In: Plant Molecular Biology, Vol. 75, No. 3, 2011, p. 263-275.

Research output: Contribution to journalArticle

Mathew, Lolita G. ; Maloney, Bryan ; Takeda, Naokazu ; Mason, Hugh. / Spurious polyadenylation of Norovirus Narita 104 capsid protein mRNA in transgenic plants. In: Plant Molecular Biology. 2011 ; Vol. 75, No. 3. pp. 263-275.
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AB - 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.

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