Identification of a discrete intermediate in the assembly disassembly of physalis mottle tymovirus through mutational analysis

Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and del etion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the asse mbly of the virus and to identify intermediates in this process. N-terminal 30, 34, 35 and 39 amino acid deletion and single C-terminal (N188) deletio n mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also co nstructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T = 3 particles in vivo while deletio ns of 34, 35 and 39 amino acid residues resulted in the mutant proteins tha t were insoluble. Interestingly, the coat protein (pR PhCP) expressed using pRSET B vector with an additional 41 amino acid residues at the N terminus also assembled into T = 3 particles that were more compact and had a small er diameter. These results demonstrate that the amino-terminal segment is f lexible and either the deletion or addition of amino acid residues at the N terminus does not affect T = 3 capsid assembly, in contrast, the deletion of even a single residue from the C terminus (PhN188 Delta 1) resulted in c apsids that were unstable. These capsids disassembled to a discrete interme diate with a sedimentation coefficent of 19.4 S. However, the replacement o f C-terminal asparagine 188 by alanine led to the formation of stable capsi ds. The C75A and D144N mutant proteins also assembled into capsids that wer e as stable as the pR PhCP, suggesting that C75A and D144 are not crucial f or the T = 3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant protein s failed to form capsids and were present as heterogeneous aggregates. Inte restingly, the pR PhK143E mutant protein behaved in a manner similar to the C-terminal deletion protein in forming unstable capsids. The intermediate with an s value of 19.4 S was the major assembly product of pR PhH69A mutan t protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188 Delta 1, pR PhH 69A and pR PhK143E mutant proteins. (C) 1999 Academic Press.