Putative glycoproteins from Brevipalpus mite- transmitted viruses and their role in the formation of the viral structure

Abstract

Citrus Leprosis (CL) is the main viral disease affecting the Brazilian Citriculture. Of multetiological origin, CL is caused by bi-segmented viruses of the genera Cilevirus [ss (+) RNA, family Kitaviridae], and Dichorhavirus [ss (-) RNA, family Rhabdoviridae], which are transmitted by mites of the genus Brevipalpus. Despite their contrasting phylogenomic, the Brevipalpus Transmitted Viruses (BTV) have a set of similar biological characteristics, probably due to a convergent evolution associated with their close relationship with the vector mites. In general, viral glycoproteins anchored in the membranes of the lipid envelope usually mediate specific recognition of virions by host cell receptors or vectors. Like other rhabdoviruses, dichorhaviruses encode a glycoprotein, the G protein, ORF5: RNA1, whose function during the replicative cycle is apparently contradictory. Although it is hypothesized that there is a correspondence between the phylogenetic relationship of the G protein of dichorhaviruses and the species of Brevipalpus mite vector, Transmission Electron Microscopy (TEM) analyzes suggest that the viral particles apparently do not have lipidic envelopes. In contrast, the P61 protein of the cileviruses interacts and reorganizes the plant endoplasmic reticulum, inducing the production of ROS (Reactive Oxygen Species) and cell death. Codified by ORFp61: RNA2 of cileviruses, in silico analyses suggest the existence of transmembrane domains and N-glycosylation sites, biochemical characteristics that indicate their possible insertion in the lipidic envelope present in virions of members of the genus Cilevirus. The main goal of the present study is to verify if the G proteins of dichorhaviruses and P61 of cileviruses are structural components of their respective virions. Therefore, the genes of both proteins will be expressed in the Baculovirus Expression Vector System (BEVS) to produce Virus-Like Particles (VPLs). The G and P61 proteins will be expressed individually or co-expressed with the matrix proteins (M) of dichorhaviruses and rabies rhabdovirus; and with P24, an integral membrane protein of cileviruses with hypothetical function similar to that of the M protein of rhabdoviruses. The capability to form VLPs (trans-complementation) with components derived from the same viral genus (homo-complementation) or chimeric VLPs combining viral proteins from different viral genera (hetero-complementation) will allow inferring about the functions of the proteins involved. Both independently expressed proteins and VLPs will be purified and used in the development of specific antisera. With them, the objective is to identify the subcellular localization of P61 and G proteins in virions during their formation and maturation processes in plant tissues and vector/host mites. It is expected to map the processes of interaction of G and P61 proteins with their host plants and vector mites, in search of the identification of possible universal or specific mechanisms of interaction, which, when blocked, may prevent viral transmission. In parallel, the generated antisera will be characterized and conveniently used as tools in the immunodetection and serotyping of BTV. (AU)