Molecular and biochemical characterization of the role of the Cucumber necrosis virus coat protein in particle structure and subcellular targeting

The Cucumber necrosis virus (CNV) particle is a T = 3 icosahedron composed of 180 identical coat protein (CP) subunits. Each subunit consists of: an RNA binding domain (R); a S domain that forms the shell and a protruding domain (P) that projects from the particle surface. The R and S domains are connected by a 34 aa arm. The aim of this study was to investigate the role of the CP N-terminal region in particle structure and subcellular targeting. Results of deletion CP mutant analyses indicate that mutants lacking the β region of the arm are capable of producing particles in infected plants; however, mutants lacking the complete arm or the ε region do not produce particles. β deletion mutant particles are less thermally stable and, under conditions where wild-type CNV particles “swell”, β(-) mutant particles disassemble, reinforcing the role of the β region in particle stability. In addition, it was found that β(-) mutant particles bind fungal zoospores less efficiently and are not fungally transmissible. GFP fusion protein constructs of CP R and arm deletion mutants were used in agro-infiltration and confocal analyses to assess the role of these regions in subcellular targeting. The studies show that specific regions in the CP N-terminus are involved in mitochondrial and chloroplast targeting. Western blot and organellar subfractionation studies show that the R domain associates with the mitochondrial inner membrane and that the C-terminal 27 aa of the arm along with the first four aa of the S domain are sufficient for targeting fusion proteins to the chloroplast stroma. In addition, a 22 aa region at the N-terminus of the S domain facilitates entry of the respective fusion proteins into the chloroplast stroma and mitochondrial matrix. The arm region of R/arm/S₂₂ GFP fusion protein associates with both mitochondria and chloroplasts; these results suggest a novel mode of dual-targeting. Together, these results demonstrate a multifunctional role for the N-terminal region of the CNV CP in virus structure and function. The possible role of subcellular targeting in virus assembly and disassembly is discussed.Land and Food Systems, Faculty ofGraduat