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

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.

Production of Pharmaceutical Proteins in Solanaceae Food Crops

“…Mathew et al [93] showed the importance of eliminating spurious polyadenylation signals within the coding sequence of the Narita 104 virus capsid protein. Such an exercise increased foreign protein expression in potato plants.…”

Design rules for efficient transgene expression in plants

“…Instability sequences avoided included AT-rich sequence strings recognized by the plant polyadenylation machinery. Spurious polyadenylation has previously been implicated in poor expression of transgenes encoding Bacillus thuringiensis toxin in potato (Haffani et al, 2000) and Norovirus Narita 104 capsid protein in tobacco (Mathew et al, 2011). In plants, the combination of sequence elements recognized as polyadenylation signals is not entirely predictable (Mathew et al, 2011).…”

“…Spurious polyadenylation has previously been implicated in poor expression of transgenes encoding Bacillus thuringiensis toxin in potato (Haffani et al, 2000) and Norovirus Narita 104 capsid protein in tobacco (Mathew et al, 2011). In plants, the combination of sequence elements recognized as polyadenylation signals is not entirely predictable (Mathew et al, 2011). The highly degenerate set that we have applied (Table 1) might be refined with further analysis, so that a computer-assisted process can converge on a solution that meets all the design criteria.…”

Norovirus Narita 104 Virus-Like Particles Expressed inNicotiana benthamianaInduce Serum and Mucosal Immune Responses

“…A plant-optimized sNaVCP gene (Genbank accession number GQ389627) from pCRblunt-sNaVCP [22] was introduced into pICH10990 (ICON Genetics, Halle, Germany) to obtain pICHsNaV. The coding sequence in pCRblunt-sNaVCP was end-tailored to create an EcoRI site at the 5′ end using a high-fidelity PCR kit (Roche) with primers sNaCP-eco (5′-GACGAATTCAACAATGAAGATGGCTTCTAATG) and M13RHT (5′-GGAAACAGCTATGACCATG).…”

“…Recombinant Norwalk virus VLP (rNV VLP) elicited VLP-specific systemic (serum IgG) and mucosal (vaginal and fecal IgA) antibodies in mice when administered orally in presence and in the absence of an adjuvant [18, 20, 21]. The plant-based expression of native NaV capsid protein was poor due to incorrect mRNA processing, but a plant optimized gene enhanced expression [22]. Also, new approaches to achieving high levels of protein using rapid virus-vectored transient expression have been developed, including the tobacco mosaic virus (TMV) system (ICON system) [23–25].…”