Expression of tobacco mosaic virus (TMV) coat protein (CP) restricts virus disassembly and alters the accumulation of the movement protein (MP). To characterize the role of structure of transgenic CP in regulating virus disassembly and production of MP, we generated CPs with mutations at residues Glu50 and Asp77, located in the interface between juxtaposed CP subunits. In transgenic Nicotiana tabacum and BY-2 cells, three categories of coat protein-mediated resistance (CP-MR) levels were identified: wild-type CP-MR; elevated CP-MR; and no CP-MR. Mutant CPs that interfered with the accumulation of virus replication complexes conferred very high levels of protection to TMV, except by CP(E50D) which provided no protection in the systemic host (Xanthi-nn) but high CP-MR in the local lesion host (Xanthi-NN). In transgenic BY-2 cells CP(E50D) strongly reduced accumulation of MP:GFP. In general, there was a strong correlation between the capacity for CP to assemble to pseudovirions and CP-MR, while there was not strong correlation with packaging viral RNA and CP-MR. The data demonstrate that interference with one or more steps in virus infection and replication by wild type and mutant CP determine the degree of CP-MR.