The Role of a C‐Terminal Seven‐Amino Acid Motif in <scp>TbCSV C3</scp> Protein and Its Interaction With <scp>NbPOLA2</scp> in Enhancing Viral Replication

The C3 protein of tobacco curly shoot virus (TbCSV), a possible evolutionary intermediate between truly monopartite begomoviruses and those requiring satellite molecules for infectivity, has been identified as a viral replication enhancer (REn). However, the mechanisms underlying this enhancement are largely unknown. In this study, we generated two mutant infectious clones of TbCSV: one with a deletion of the 3' end region of the C3 gene that does not overlap with C2 (TbCSV_dC3) and another in which this region was replaced by a phylogenetically unrelated iLOV gene sequence (TbCSV_dC3-iLOV). Our findings highlight the crucial role of the 3' end region of C3 for viral DNA accumulation and further demonstrated that overexpression of TbCSV C3 protein in trans complements the functional deficiency of TbCSV_dC3. Further analyses revealed the essential role of the C-terminal seven-amino acid motif from residues 123-129 of C3 in replication enhancement. Previous studies suggested that both intra- and intermolecular interactions of C3/AC3 proteins encoded by some other geminiviruses are vital for their capacity to enhance replication. Interestingly, among the tested potential interactors, NbPOLA2, a subunit of DNA polymerase α, was confirmed to interact with C3 in yeast and in planta. Our findings indicated that NbPOLA2 positively regulates TbCSV replication and infection and that the seven-amino acid motif (residues 123-129) in C3 is required for recruiting NbPOLA2 to facilitate TbCSV replication by mediating the viral double-stranded DNA (dsDNA) replication intermediate synthesis. These findings contribute to our understanding of the mechanisms through which the C3 protein enhances TbCSV replication.