Cilevirus and dichorhavirus glycoproteins target overlapping host proteins in the Brevipalpus yothersi vector

Enveloped plant viruses are rare, and accumulating evidence suggests deep evolutionary ties to arthropod hosts. Brevipalpus-transmitted viruses (BTVs), which include cileviruses, higreviruses, and dichorhaviruses, are transmitted exclusively by Brevipalpus mites, causing localized infections in plants. Some cileviruses and dichorhaviruses can also replicate within vector cells. Despite their enveloped virions, the involvement of virion structural proteins mediating the interaction with the vector remains unresolved. Here, we investigated the putative glycoproteins P61 of citrus leprosis virus C (CiLV-C; Cilevirus, Kitaviridae) and G of clerodendrum chlorotic spot virus (ClCSV; Dichorhavirus, Rhabdoviridae) to define their interaction networks in the Brevipalpus yothersi vector. Using membrane-based yeast two-hybrid screening with a B. yothersi cDNA library, we identified 73 interactors for P61 and 162 for G, including a shared core set enriched for membrane-associated proteins involved in intracellular trafficking, ER-Golgi dynamics, protein synthesis and folding, and signal transduction. Numerous hypothetical membrane proteins emerged as strong candidates for viral receptors or co-receptors. Three proteins (ARF1, SERP2, and a hypothetical transmembrane protein) were validated by BiFC and Co-IP in Spodoptera frugiperda (Sf9) cells, confirming interactions with both viral proteins in an arthropod-like environment. Together, this work provides the first comprehensive interactome of BTV glyco-like proteins with those of its natural vector and reveals partially convergent host-interaction strategies between phylogenetically distinct viruses, establishing a mechanistic basis for dissecting BTV transmission. IMPORTANCEBTVs damage economically important crops worldwide, and citrus leprosis (CL) is a significant disease in the Brazilian citrus belt. Brazil is responsible for over 75% of global sweet orange juice production and widely cited estimates indicate that CL causes >US$69 million in annual yield losses and acaricide-based control of Brevipalpus yothersi, the vector of CiLV-C and several other BTVs. Despite this impact, the molecular basis of BTV acquisition, persistence, and transmission by Brevipalpus spp. remains largely unexplored. Here, we provide the first systematic map of mite proteins interacting with glycoproteins from two BTVs belonging to distinct viral families. These interactomes reveal key components of the cellular machinery targeted by cileviruses and dichorhaviruses in their arthropod vector, offering mechanistic insight into how these viruses establish and maintain infections in mites. This work establishes a foundational framework for future studies aimed at disrupting BTV transmission and developing sustainable control strategies.