Identification of Brevipalpus yothersi receptors of the cile- and dichorha-virus glycoproteins

• Grant number: 23/09518-4
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: November 30, 2023
• End date: November 29, 2024
• Field of knowledge: Agronomical Sciences - Agronomy - Plant Health
• Principal Investigator: Elliot Watanabe Kitajima
• Grantee: Camila Chabi de Jesus
• Supervisor: Anna Elizabeth Whitfield
• Host Institution: Escola Superior de Agricultura Luiz de Queiroz (ESALQ). Universidade de São Paulo (USP). Piracicaba , SP, Brazil
• Institution abroad: North Carolina State University (NC State), United States
• Associated to the scholarship: 20/15413-2 - Putative glycoproteins from Brevipalpus mite- transmitted viruses and their role in the formation of the viral structure, BP.PD

Abstract

Glycoproteins are essential structural components of enveloped viruses and have a critical role in viral pathogenesis. Viruses transmitted by mites of the genus Brevipalpus belong to three distinct genera: Cilevirus and Higrevirus [ss(+)RNA genome, family Kitaviridae], and Dichorhavirus [ss(-)RNA genome, family Rhabdoviridae]. Virions of Brevipalpus-transmitted viruses (BTV) of the genus Cilevirus are enveloped, whereas different from other rhabdoviruses, dichorhaviruses, which encode typical glycoproteins, appear to be naked viruses. In cileviruses and dichorhaviruses, the identity and role of structural proteins are poorly understood. In silico and transient expression analyses indicate that the P61 of cileviruses (ORF_p61:RNA2) and the G of dichorhaviruses (ORF_G:RNA1): (i) are glycosylated; (ii) show transmembrane domain and signal peptide; and (iii) have membrane-associated cellular localization; therefore, P61 and G may act as viral glycoproteins. In Arabidopsis, P61 of the cilevirus citrus leprosis virus C (CiLV-C) colocalizes and remodels the endoplasmic reticulum and the Golgi complex, and induces ROS production, leading to the production of necrotic symptoms observed during the infections caused by BTV. Thus, P61-CiLV-C seems to play an important role in the interaction with its hosts. CiLV-C and the dichorhavirus clerodendrum chlorotic spot virus (ClCSV) are transmitted by B. yothersi. Because it is a rhabdovirus, the glycoprotein of ClCSV has phylogenetic and physicochemical characteristics comparable to other animal and plant rhabdoviruses that are widely known. Globally, we hypothesize that P61-CiLV-C and G-ClCSV are required for invasion, infection, and spread in B. yothersi vector. To confirm the participation of these proteins as structural components of BTV virions, the applicant's research project in Brazil aims to express P61 and G proteins using a baculovirus-insect cell expression system. In parallel, this BEPE project proposes to identify candidate receptors in the Brevipalpus yothersi vector that physically interact with P61-CiLV-C and G- ClCSV using split ubiquitin membrane-based yeast two-hybrid system. This project will be conducted under the supervision of Dr. Anna Whitfield at the Plant-Virus-Vector Interactions Lab (North Carolina State University, Raleigh, USA). Dr. Whitfield is an expert on the plant-virus-vector interactions at the molecular level and has several studies on the interaction of the corn planthopper (Peregrinus maidis) and the alphanucleorhabdovirus maize mosaic virus. To achieve the objectives of the current project, a cDNA library will be prepared from 200 non-viruliferous B. yothersi mites, which will be enriched, digested, and cloned into yeast prey plasmids. Genes p61 and G will be cloned into the yeast bait plasmids. The cloning procedures, sequence confirmation, yeast transformation, and positive selection of the prey-bait plasmids interactions will be performed according to the Dualsystems protocol (Dualsystems Biotech, Schlieren, Switzerland). The predicted amino acid sequences of the tentative preys will be analyzed for the identification of functional motifs related to cytoplasmic and/or nuclear membrane receptors and other functions e.g. immune response, protein folding, trafficking, and cell signaling. The interactomics networks of BTV-studied proteins will be described using bioinformatic tools. If possible, other assays for protein-protein interaction (co-immunoprecipitation and co-localization) will be performed to confirm the protein-protein interaction in insect cells. With the development of this project, it will be possible for the first time to identify molecular partners of BTV in Brevipalpus mites. These results may corroborate the hypotheses on the function of the glycoproteins of cile- and dichorha-viruses, and might provide new approaches for the development of biotechnological products intended to attenuate the vectorial activity of Brevipalpus mites. (AU)