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Identification of a discrete intermediate in the assembly/disassembly of physalis mottle tymovirus through mutational
M Sastri1, D S Reddy, S S Krishna
1Department of Biochemistry, Indian Institute of Science, Bangalore, 560 012, India.
Abstract:
Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and deletion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the assembly 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) deletion mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also constructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T=3 particles in vivo while deletions of 34, 35 and 39 amino acid residues resulted in the mutant proteins that 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 smaller diameter. These results demonstrate that the amino-terminal segment is flexible 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 (PhN188Delta1) resulted in capsids that were unstable. These capsids disassembled to a discrete intermediate with a sedimentation coefficent of 19.4 S. However, the replacement of C-terminal asparagine 188 by alanine led to the formation of stable capsids. The C75A and D144N mutant proteins also assembled into capsids that were as stable as the pR PhCP, suggesting that C75 and D144 are not crucial for the T=3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant proteins failed to form capsids and were present as heterogeneous aggregates. Interestingly, 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 mutant protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188Delta1, pR PhH69A and pR PhK143E mutant proteins.
Insights
Physalis mottle tymovirus (PhMV) coat protein mutants reveal that N-terminal flexibility is key for T=3 capsid assembly, while C-terminal deletions cause instability and identify a 19.4 S assembly intermediate.
Area of Science:
- Virology
- Structural Biology
- Protein Biochemistry
Background:
- Icosahedral virus assembly intermediates are typically transient and challenging to isolate.
- Understanding viral capsid formation is crucial for developing antiviral strategies.
Purpose of the Study:
- To investigate the role of specific amino acid residues in physalis mottle tymovirus (PhMV) coat protein assembly.
- To identify and characterize transient assembly intermediates during PhMV formation.
Main Methods:
- Construction and expression of N-terminal and C-terminal deletion mutants of PhMV coat protein in E. coli.
- Creation and expression of site-specific mutants (H69A, C75A, W96A, D144N, K143E, etc.).
- Analysis of self-assembly properties, particle stability, and intermediate formation using sedimentation analysis.
Main Results:
- N-terminal deletions/additions (up to 30 residues) did not impede T=3 capsid assembly, indicating N-terminal flexibility.
- C-terminal single residue deletion (PhN188Δ1) resulted in unstable capsids that disassembled into a 19.4 S intermediate.
- Mutants pR PhW96A and pR PhD144N-T151A failed to form capsids, forming heterogeneous aggregates, while pR PhK143E and pR PhH69A showed instability and intermediate formation.
Conclusions:
- The N-terminus of PhMV coat protein is flexible and tolerant to modifications regarding T=3 capsid assembly.
- The C-terminus is critical for capsid stability, and its deletion leads to disassembly and a distinct 19.4 S intermediate.
- Specific residues (H69, K143) and potential interactions involving W96 and D144/T151 are important for proper capsid formation or stability.
