Localization of protein P61 of cileviruses by immunochemical methods

• Grant number: 25/03469-7
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: May 01, 2025
• End date: March 31, 2026
• Field of knowledge: Agronomical Sciences - Agronomy - Plant Health
• Principal Investigator: Juliana de Freitas Astúa
• Grantee: Thaís Teodoro de Oliveira Santos
• Host Institution: Instituto Biológico (IB). Agência Paulista de Tecnologia dos Agronegócios (APTA). Secretaria de Agricultura e Abastecimento (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 19/25078-9 - Genomics and trascriptome of virus-vector-host plant relationship in the pathosytem of Brevipalpus transmitted viruses; systematic and evolution of Brevipalpus mites and their endosymbionts; new strategies to manage citrus Leprosis in the S. Paulo State, AP.TEM

Abstract

The objective of this work is to detect the presumptive glycoprotein p61 as a structural componente of the mature virions of cileviruses. The real composition of cilevirus virions is still nuclear. In silico evaluation suggests that proteins p61 and p24 are endowed with transmembrane domains which allow them to be inserted in host cell membranes and in virion's envelope. However, there is experimental proof of the insertion of p24 in cell membranes. The presente Project intends to identify p61 protein in the virion's envelope, either in infected plant cells as in viruliferous mite vector, Brevipalpus yothersi, by immuno-histochemistry and transmission eléctron microscopy. The expression. of native p61 protein in Sf9 insect cells, based on a baculovirus system, results in the premature death of these cells. Thus the amount of recovered p61 protein is very low and insuficiente for a immunization program in rabbit to produce specific antibodies. The cell death process noticed in transfected insect cells is observed also during ectopic expression. of this protein in the assay plant Nicotiana benthamiana. To by-pass protein expression., an alternative is the synthesis of peptides, which would be used for polyclonal antibodies production. In silico analysis based on bioinformatic techniques, p.ex., 3D modelling of proteins and immunoreactive profiles, would permit evaluate the peptides corresponding to the exposed and immunogenic regions of the protein p61, from stranis of the citrus leprosis virus C (CiLV-1c, Cilevirus leprosis) and the homologous proteins from other cilevirus species. Generated antibodies would be used in in situ immunolocalization, after being conjugated with coloidal gold particles.