Spurious polyadenylation of Norovirus Narita 104 capsid protein
mRNA in transgenic plants
Lolita G. Mathew
•
Bryan Maloney
•
Naokazu Takeda
•
Hugh S. Mason
Received: 23 May 2010 / Accepted: 22 December 2010 / Published online: 4 January 2011
Ó Springer Science+Business Media B.V. 2011
Abstract Noroviruses are members of the family Calici-
viridae, 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 expres-
sion 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 polyadenyl-
ation. 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 iden-
tify 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 Geno-
group I Norovirus) in transiently transfected plants. These
findings provide evidence for impaired expression of NaV-
CP in transgenic plants mediated by spurious polyadenyla-
tion signals, and demonstrate the need to scrupulously search
for potential polyadenylation signals in order to improve
transgene expression in plants.
Keywords Polyadenylation Á Norovirus Á Plant based
vaccine Á Plant viral vectors Á Transgenic plants
Introduction
Biotechnology has played a major role in advancing the
quality of life by enabling expression of valuable proteins
in heterologous expression systems. Expression of recom-
binant proteins in plants has many potential applications,
including production of vaccine antigens. However, poor
expression of the protein of interest in plants can be a
limiting factor. To improve expression of foreign genes in
plants, studies have examined the mRNA 5
0
and 3
0
untranslated regions (UTR), and the use of a variety of
Electronic supplementary material The online version of this
article (doi:10.1007/s11103-010-9725-1) contains supplementary
material, which is available to authorized users.
L. G. Mathew Á H. S. Mason (&)
Center for Infectious Diseases and Vaccinology (CIDV),
The Biodesign Institute at Arizona State University,
1001 South McAllister Avenue, Tempe, AZ 85287-5401, USA
e-mail: hugh.mason@asu.edu
L. G. Mathew Á H. S. Mason
The School of Life Sciences, Arizona State University,
Tempe, AZ 85287-5401, USA
Present Address:
L. G. Mathew
USDA-ARS Arid Lands Agricultural Research Center,
21881 North Cardon Lane, Maricopa, AZ 85138, USA
N. Takeda
Department of Medical Sciences, Ministry of Public Health,
Research Collaboration Center on Emerging and Re-emerging
Infections, National Institute of Health, Building 10,
Tivanond 14 Road, Muang, Nonthaburi 11000, Thailand
B. Maloney
Boyce Thompson Institute for Plant Research, Tower Road,
Ithaca, NY 14853, USA
123
Plant Mol Biol (2011) 75:263–275
DOI 10.1007/s11103-010-9725-1