Intra-viral protein-protein interactions could expand the functional portfolio of C4 in okra-infecting geminiviruses

Viruses must rewire the cells they infect to promote their own multiplication, a task they accomplish through the deployment of multifunctional virus-encoded proteins. However, the limited coding capacity imposed by small viral genomes constrains their functional repertoire. Recently, it has been proposed that proteins encoded by plant-infecting geminiviruses can physically interact with one another, forming intra-viral protein complexes that may acquire novel activities and thereby expand viral functionality. Here, we test this concept by focusing on the C4 proteins of two okra-infecting geminiviruses, bhendi yellow vein mosaic virus (BYVMV) and okra enation leaf curl virus (OELCuV). We systematically assess whether these C4 proteins associate with cognate virus-encoded proteins or with proteins encoded by co-infecting DNA satellites using yeast two-hybrid, detecting direct protein-protein interactions; co-immunoprecipitation, identifying proteins associated within the same complex; and bimolecular fluorescence complementation, reporting close association of proteins within living cells. These experimental approaches were complemented by AlphaFold-based predictions of direct protein-protein interactions. Our results indicate that C4 proteins can associate to multiple viral proteins. Notably, the subcellular localization of intra-viral protein complexes appears to be largely conserved across different geminiviruses. Taken together, our findings suggest that C4 proteins may perform additional functions in infected cells beyond those described for individual proteins, highlighting the importance of studying viral protein function within the context of infection.