Two Cassava Novel Cap-Binding Proteins Interact with VPgs of Cassava Brown Streak Virus and Ugandan Cassava Brown Streak Virus

Cassava (Manihot esculenta) is an important tropical subsistence crop that is affected by cassava brown streak disease (CBSD) in Sub-Saharan Africa. CBSD is caused by two distinct virus species: Cassava brown streak virus (CBSV) and Ugandan Cassava brown streak virus (UCBSV). Both are whitefly-transmitted ipomoviruses with a (+)-sense single-stranded RNA genome of about 9 kb. Covalently linked to the 5’-terminus of the viral genomic RNA is a genome-linked viral protein (VPg), which is functionally analogous to the cap structure of mRNA and serves as the binding site for eukaryotic translation initiation factor 4E (eIF4E and eIF(iso)4E). This interaction is a prerequisite for translation of viral genomic RNA in host cells. We identified and cloned five cassava eIF4E genes, Cassava 4.1_016601m, 016620m, 013223m, 013732m, and 015501m, and assayed the interaction between the eIF4E proteins and the VPgs of CBSV and UCBSV. Two novel cap-binding proteins (nCBPs, a subfamily of eIF4E proteins), 015501m and 013732m, were shown to have positive interactions with both CBSV and UCBSV VPgs. Furthermore we found that the full-length nCBPs are essential for the eIF4E-VPg interaction, as N- or C- terminal deletion decreases this interaction dramatically. We also identified several key amino acids in the nCBPs contributing to the eIF4E-VPg interaction. Our results indicate that P99 and S161 in 013732m and the corresponding P104 and S166 in 015501m may play crucial roles in the eIF4E-VPg interaction, as either single or double mutations of these amino acids partially or completely destroyed their interaction with CBSV and UCBSV VPg.