A new approach for super-expression of the influenza virus epitope M2e in plants has been developed on the basis of a recombinant Tobacco mosaic virus (TMV, strain U1) genome designed for Agrobacterium-mediated delivery into the plant cell nucleus. The TMV coat protein (CP) served as a carrier and three versions of the M2e sequence were inserted into the surface loop between amino acid residues 155 and 156. Cysteine residues in the heterologous peptide were thought likely to impede efficient assembly of chimeric particles. Therefore, viral vectors TMV-M2e-ala and TMV-M2e-ser were constructed in which cysteine codons 17 and 19 of the M2e epitope were substituted by codons for serine or alanine. Agroinfiltration experiments proved that the chimeric viruses were capable of systemically infecting Nicotiana benthamiana plants. Antisera raised against TMV-M2e-ala virions appear to contain far more antibodies specific to influenza virus M2e than those specific to TMV carrier particle (ratio 5:1). Immunogold electron microscopy showed that the 2-epitopes were uniformly distributed and tightly packed on the surface of the chimeric TMV virions. Apparently, the majority of the TMV CP-specific epitopes in the chimeric TMV-M2e particles are hidden from the immune system by the M2e epitopes exposed on the particle surface. The profile of IgG subclasses after immunization of mice with TMV-M2e-ser and TMV-M2e-ala was evaluated. Immunization with TMV-M2e-ala induced a significant difference between the levels of IgG1 and IgG2a (IgG1/IgG2a=3.2). Mice immunized with the chimeric viruses were resistant to five lethal doses (LD50) of the homologous influenza virus strain, A/PR/8/34 (H1N1) and TMV-M2e-ala also gave partial protection (5LD50, 70% of survival rate) against a heterologous strain influenza A/California/04/2009 (H1N1) (4 amino acid changes in M2e). These results indicate that a new generation candidate universal nanovaccine against influenza based on a recombinant TMV construct has been obtained.

We report that unprocessed tobacco pectin methylesterase (PME) contains N-terminal pro-sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro-sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro-sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting.

Co-agroinjection of Nicotiana benthamiana leaves with the pectin methylesterase (proPME) gene and the TMV:GFP vector resulted in a stimulation of virus-induced RNA silencing (inhibition of GFP production, virus RNA degradation, stimulation of siRNAs production). Conversely, coexpression of TMV:GFP with either antisense PME construct or with enzymatically inactive proPME restored synthesis of viral RNA. Furthermore, expression of proPME enhanced the GFP transgene-induced gene silencing accompanied by relocation of the DCL1 protein from nucleus to the cytoplasm and activation of siRNAs and miRNAs production. It was hypothesized that DCL1 relocated to the cytoplasm may use as substrates both miRNA precursor and viral RNA. The capacity for enhancing the RNA silencing is a novel function for the polyfunctional PME.

The understanding of genetic diversity, geographic distribution, and antigenic properties of Plum pox virus (PPV) is a prerequisite to improve control of sharka, the most detrimental viral disease of stone fruit crops worldwide. Forty new PPV strain C isolates were detected in sour cherry (Prunus cerasus) from three geographically distant (700–1100 km) regions of European Russia. Analysis of their 3’-terminal genomic sequences showed that nineteen isolates (47.5%) bear the D96E mutation in the universal epitope of the coat protein. Almost all of them cannot be detected by the monoclonal antibody 5B in triple antibody sandwich enzyme-linked immunosorbent assay and Western blot analysis that may potentially compromise serological PPV detection in cherries. Full-length genomes of seven PPV-C isolates were determined employing next-generation sequencing. Using the Recombination Detection Program (RDP4), the recombination event covering the region from (Cter)P1 to the middle of the HcPro gene was predicted in all the available PPV-C complete genomes. The isolates Tat-4, belonging to the strain CV, and RU-17sc (PPV-CR) were inferred as major and minor parents, respectively, suggesting possible pathways of evolution of the cherry-adapted strains. Downy cherry (P. tomentosa) was identified as the natural PPV-C host for the first time.

Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV) genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP) gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV) did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV‑based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod‑shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP) described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.