Expression of Hemagglutinin-Neuraminidase glycoprotein of Newcastle disease virus in agroinfiltrated Nicotiana benthamiana plants

EVANGELINA GÓMEZ; SILVINA CHIMENO ZOTH; ELISA CARRILLO; ANALÍA BERINSTEIN

Verona, Italia

Congreso; Plant-Based Vaccines and Antibodies 2007 Conference; 2007

Newcastle disease virus (NDV) is a highly contagious viral disease distributed worldwide that infects a wide range of both domestic and wild birds causing serious economic losses in the poultry industry. It is an enveloped virus belonging to the Paramixoviridae family which major antigens are Hemagglutinin-Neuraminidase (HN) and Fusion surface glycoproteins. Both proteins have been employed as targets for structure-based drugs and subunit vaccines design in an attempt to provide alternative strategies to control the disease without handle infectious virus. We have already described the use of transgenic potato plants as oral immunogens against NDV. Those plants expressed the HN glycoprotein under the regulation of CaMV 35S promoter with a very low performance. The main objective of the work presented here was to enhance the expression of the HN glycoprotein of NDV through a protein-directing strategy and a promoter change. The enhanced expression would allow obtaining a plant-based vaccine for susceptible hosts. A 1.7 kb fragment encompassing the coding region of the viral hemagglutinin- neuraminidase protein was cloned into a plant expression cassette under the rubisco small subunit promoter. Four constructs were generated: (1) pBinHN, (2) pBinSP/HN, (3) pBinSP/HN/KDEL, (4) pBinHN/KDEL, where SP is the plant signal peptide and KDEL the endoplasmic reticulum (ER) retention sequence. Another construct was generated in which the HN was cloned without its own signal peptide: pBinSP/-HN/KDEL. In all cases the protein remained fused to a c-myc tag. The resulted binary vectors were electroporated in Agrobacterium tumefaciens GV 3101. The recombinant bacteria were grown up for 48 h in the presence of the corresponding antibiotics. The gene virulence was induced for 5 h prior to infiltrate Nicotiana benthamiana leaves. Agrobacteria was inoculated at an OD600= 1 and leaves were harvested 48 h later. A green fluorescent protein (GFP) construct was used as agroinfiltration positive control. Leaves were homogeneized with liquid N2 and total proteins were extracted with an appropriate buffer. Protein samples were subjected to SDS-PAGE under reducing conditions and then Western blot analysis were carried out using a) an anti-c-myc monoclonal antibody (SIGMA) to visualize the fusion proteins and b) anti-GFP polyclonal antibodies to visualize GFP. A standard hemagglutination assay was performed to confirm hemagglutination activiy of plant-derived proteins. Western blot assays showed the presence of the HN protein. This result was obtained in all cases where the constructs contained just one signal peptide and revealed that the protein was being sent to the ER by both the viral and, as expected, the plant signal peptide, indicating that it was further undergoing post-translational modifications. Notably, no protein was obtained when the two signal peptides were present in the constructions. pBinHN derived plant extract rendered less protein compared with pBinHN/KDEL and pBinSP/-HN/KDEL. This could be so because the protein obtained through the pBinHN construct was directed to the ER but it followed a secretory pathway to the apoplast where the turnover of proteins is higher. All plant-derived HN proteins showed hemagglutination activity. Our results demonstrate that it is possible to obtain the HN protein in a transient expression assay and that the viral signal peptide that directs the protein to the ER in avian cells also targets the protein to the ER of transformed leaves efficiently and the protein obtained retains the hemagglutination activity. We have been able to enhance the accumulation levels by targeting to and retaining the protein in the ER.