Noemi Cerovska

The detection of recombinant, tuber necrosing isolates of Potato virus Y (PVYNTN) using a three-primer PCR based in the coat protein gene

A simple and reliable procedure for reverse transcription-polymerase chain reaction (RT-PCR) detection and strain differentiation of Potato virus Y (PVY) was developed. Three primers were designed within the coat protein (CP) and nuclear inclusion protein b (NIb) region, exploiting a single base polymorphism identified as being present in all the recombinant PVY(NTN) isolates published. Samples infected with PVY produce a single band of 569 bp, while isolates belonging to PVY(NTN) strain give an additional band of 334 bp. The technique was tested on a collection of well-characterised isolates of PVY from a range of strains and was found to detect all of the isolates reported as belonging to the PVY(NTN) strain. All of the isolates detected possess a recombination event within the coat protein. Further sequence analysis revealed that all the recombinant PVY(NTN) isolates reported thus far would be detected using this assay, whilst isolates thought to be PVY(NTN) that do not possess the coat protein recombination event would not be detected.

Short communication: Molecular analysis of Potato leafroll virus isolates from the Czech Republic.

The complete genomes of three Czech isolates VIRUBRA 1/045, VIRUBRA 1/046, and VIRUBRA 1/047 of Potato leafroll virus (PLRV) were sequenced and compared with 13 complete sequences of PLRV isolates available in GenBank. Among the Czech isolates, VIRUBRA 1/046 and 1/047 showed the highest nucleotide (nt) identity (98.7%). PLRV was the most conserved virus in both open reading frames (ORFs) 3 and 4. The most variable regions were ORFs 0 and Rap1. Interestingly, isolate VIRUBRA 1/045 significantly differed from the other two Czech isolates in ORFs 0 and 1. Moreover, we identified mutations in the amino acid (aa) sequences, which were specific for the Czech isolates. Phylogenetic analysis based on ORF0 showed that the Czech isolates could be classified in two of the three groupings of the phylogenetic tree obtained. This is the first report on sequence analysis of the genome sequences of PLRV isolates from the Czech Republic.

Plant viruses as scaffolds for the presentation of vaccine epitopes

Within the last two decades, plant viral vectors have emerged as an excellent tool for the expression of foreign peptides and proteins. Virus particles carrying foreign antigenic epitopes present some interesting advantages for vaccine design and other applications. This review covers recent advances in the use of some typical plant viruses with helical particles that present heterologous peptides with particular emphasis on particles derived from the Potato virus X (PVX) and its uses.

Efficient expression of Human papillomavirus 16 E7 oncoprotein fused to C-terminus of Tobacco mosaic virus (TMV) coat protein using molecular chaperones in Escherichia coli.

The Human papillomavirus 16 (HPV16) E7 oncoprotein is a promising candidate for development of anti-cancer therapeutic vaccine. We have prepared the expression construct carrying mutagenized E7 oncoprotein fused to the C-terminus of Tobacco mosaic virus (TMV) coat protein via 15 amino acids β-sheet linker. The fusion protein was expressed in Escherichia coli MC 1061 cells. We have obtained high level expression, but most of the protein remained in insoluble inclusion bodies. To increase the ratio of soluble protein various molecular chaperones (TF, DnaK-DnaJ-GrpE, GroEL-GroES) were used. The immunological reactivity of expressed recombinant protein was evaluated with anti-E7 and anti-TMV antibodies. The distribution of expressed product during ultracentrifugation on sucrose gradient was studied.

Potato virus X displaying the E7 peptide derived from human papillomavirus type 16: a novel position for epitope presentation

AbstractPotato virus X (PVX) is widely used as a peptide presentation system in plant biotechnology, mostly by fusion of desired peptides to the N-terminus of its capsid protein (CP). Considering that some epitopes can interfere with the stability and/or self-assembly of PVX CP when fused to its N-terminus, we evaluated four other possible sites for fusion using the E7 epitope derived from human papillomavirus type 16 with different tags. We prepared eight different PVX CP constructs modified with the E7 epitope fused with the 6xHis tag in both orientations (6xHis-E7, E7-6xHis), cloned them into the PVX-based vector pGR106 and expressed them transiently in Nicotiana benthamina plants. Only the fusion site located after amino acid 23 led to systemic infection of plants and the production of recombinant proteins, but no viral particles were detected. When we replaced the 6xHis with StrepII tag, the modified virus infected plants systemically, expressed proteins assembled into viral particles and the epitopes were located on the particle surface. The results of this study indicate that the new position within the PVX CP can be used for peptide presentation on the surface of PVX particles and is promising for PVX based production of therapeutic compounds in plants.

Expression of a recombinant Human papillomavirus 16 E6GT oncoprotein fused to N- and C-termini of Potato virus X coat protein in Nicotiana benthamiana

Infection with Human papillomaviruses (HPVs) is linked to cervical cancer, which is one of the most common cancers found among women worldwide. Despite preventive immunization, a therapeutic vaccine, targeting infected individuals, is required. Vaccine strategies for treatment of HPV—induced cervical cancer are based on E7 and E6 oncoproteins. In this work we report the transient expression of chimeric particles containing the E6 oncoprotein from Human papillomavirus type 16 (HPV-16) in plants. We fused a mutagenized coding sequence of the E6 oncoprotein (E6GT) with the coding sequence of Potato virus X coat protein (PVX CP) both with the 5′- and 3′-terminus using linkers of different length (0, 4 or 15 amino acids). The expression in E. coli was performed to assess the characteristics of the recombinant protein prior to plant expression. The yield and immunological reactivity of the expressed proteins were screened with anti-PVX CP and E6 antibodies. The highest yields of chimeric particles were observed in the transgenic N. benthamiana expressing Potato virus A HC-Pro protein and the Tobacco mosaic virus movement protein. When inoculated on host plants, these recombinant viruses were not able to spread systemically. The obtained results revealed the new relations for design of expression cassettes for plant-based vaccine production.

The influence of the N- and C- terminal modifications of Potato virus X coat protein on virus properties

The Potato virus X (PVX)-based vector was used for the construction of N- and C-terminally modified PVX coat protein (XCP) chimeras. N-terminal XCP modifications do not influence the viral life cycle, whereas the simple XCP C-terminal fusion impedes the viral replication. We designed several C-terminally modified XCP chimeras and tested their viabilities in various Nicotiana benthamiana genotypes. Our results showed the negative impact of 3′-terminal modification of XCP on the chimera’s life cycle. To ensure chimeric constructs stability, the second copy of the last 60 nucleotides of XCP followed by the 3′-untranslated region (UTR) was added downstream of the recombinant sequence. Simultaneously, the first copy of the last 60 nucleotides of XCP was mutated in order to prevent recombination between the two identical sequences. The movement protein of Tobacco mosaic virus expressed in transgenic N. benthamiana plants positively affected the cell-to-cell spread of C-terminally modified XCP chimeras.

Potato virus X induces DNA damage in leaf nuclei of the host plant Nicotiana tabacum L. var. xanthi

We employed the comet assay (single cell gel electrophoresis) to evaluate induced DNA damage in nuclei isolated from tobacco leaves (Nicotiana tabacum var. xanthi) inoculated with Potato virus X (PVX). The highest DNA damage, expressed by the tail moment value, was observed in the inoculated leaves and decreased in the 1st to 4th systemic leaves. DNA damage increased with the time after the inoculation (from day 3 to day 21) and was higher in nuclei isolated from a part of the leaf at the petiole compared to nuclei isolated from the leaf tip. A Pearson moment correlation (r = 0.94) between the induced DNA damage and the PVX titres expressed by ELISA absorbance values was observed. The PVX infection did not induce a significant increase in the rate of somatic mutations evaluated by appearance of dark green, yellow, and double green/yellow sectors on the heterozygous pale green leaves of N. tabacum var. xanthi.

Transgenic tobacco plants carrying the non-structural P3 gene of potato virus A

Transgenic tobacco (Nicotiana tobacum) plants carrying the gene coding for potato virus A (PVA) non-structural P3 protein were prepared by inoculation with Agrobacterium tumefaciens. Seeds from self-pollinated flowers (T1 generation) were collected. To estimate the effectiveness of vertical transfer of the introduced gene and usefulness of respective plant lines for further experiments, the T1 generation was characterized by testing its ability to grow in the presence of kanamycin (Km) and by PCR of both neomycin phosphotransferase (nptII) and PVA P3 genes. Eight and ten of 29 lines showed Mendelian segregation of Km-resistant phenotype 3:1 and ≥15:1, respectively, the T1 of eleven lines showed low Km resistance. Selected PCR-positive lines were tested for the presence of P3 mRNA. In most cases the transgene transcription was dependent on the presence or absence of Km in the plant growth medium. Prepared transgenic plants were furthermore tested for sensitivity to PVA and potato virus Y (PVY) infection. All of them showed identical symptom development as the non-transgenic control plants.

New positions for peptide presentation in Potato virus X capsid protein

Abstract Potato virus X (PVX) is widely used as a peptide presentation system in plant biotechnology, mostly for transient expression of desired peptides fused to the N-terminus of its capsid protein (CP). Here we describe testing/investigation of new positions for peptide presentation based on seven putative surface loops of PVX CP. We performed bacterial expression of fourteen different PVX CPs modified by the insertion of the epitope derived from the E7 oncoprotein (E7 epitope) of Human papillomavirus type 16 fused with His tag. The expression from vector pMPM-A4Ω in Escherichia coli MC1061 was performed to evaluate the capacity of the PVX CP platform to tolerate the insertion of E7 epitope fused with His tag in the seven putative surface loops. The immunological characteristics of expressed epitopes were assessed by Western blot using both anti-PVX CP and anti-His antibodies and by immunoelectron microscopy.