CARBOXYLATE INTERACTIONS INVOLVED IN THE DISASSEMBLY OF TOBACCO MOSAIC TOBAMOVIRUS

Structural studies of tobacco mosaic tobamovirus (TMV) have identified two coat protein (CP) intersubunit carboxyl-carboxylate interactions and one CP carboxylate-RNA phosphate interaction whose electrostatic r epulsion is believed to drive virion disassembly. In this study, the i nvolvement of each interaction in the disassembly process was examined . Site-directed mutagenesis was used to replace selected negatively ch arged CP residues, E or D, with neutral residues, Q or N, respectively . Purified mutant CPs were assayed for their ability to inhibit wild-t ype TMV disassembly both in vitro and in vivo. Results indicate that t he lateral carboxylate interaction made by residue E106 is much more c omplex than previously thought, involving three residues, E95, E97, an d D109, from an adjacent subunit. Mutations at all three residues are required to inhibit disassembly significantly Different mutant coat pr oteins inhibited disassembly of the wild-type virus to varying degrees . Mutant E50Q, which modified the axial intersubunit interaction, had the greatest ability to inhibit disassembly followed by mutants E95Q/E 97Q/D109N and D116N, which modified the lateral and CP-RNA interaction s, respectively. Within each set of interacting carboxylate groups, mu tations in the face opposite the disassembling surface of the TMV viri on conferred the greatest ability to inhibit disassembly. This observa tion is consistent with the polar nature of TMV disassembly and confir ms that repulsive intersubunit interactions derived from the 5' termin al subunits provide the key controlling mechanisms for virion disassem bly. (C) 1996 Academic Press, Inc.