Role of interfacial residues in the subunit folding and assembly of physalis mottle tymovirus

Abstract

Molecular Mechanism of Assembly of Physalis Mottle Virus (PhMV) and Its Application as an Antigen Presentation System

Abstract and Synopsis Introduction Virus assembly is a critical step in the viral life cycle, involving precise protein-protein and protein-nucleic acid interactions. Plant viruses, with their ordered and symmetric capsids, serve as excellent models to study macromolecular assembly. They are also useful as epitope presentation systems for vaccine development. This thesis focuses on the assembly mechanism of Physalis mottle virus (PhMV) and its potential as a carrier for foreign epitopes.

PhMV Structure and Assembly

PhMV is a small spherical plant virus with a positive-sense ssRNA genome (6.67 kb) encapsidated in 180 identical coat protein (CP) subunits arranged in T=3 icosahedral symmetry.

Empty capsids form both in vivo and in vitro, suggesting that assembly information resides within the primary structure of CP subunits.

Recombinant CP (rCP) expressed in E. coli self-assembled into virus-like particles (VLPs), enabling mutational analysis.

Objectives

Identify residues involved in inter-subunit interactions.

Perform site-directed mutagenesis of these residues.

Characterize mutant CPs for folding, assembly, and intermediates.

Delineate the assembly pathway.

Identify suitable sites on PhMV VLPs for foreign epitope fusion.

Use PhMV VLPs to present epitopes from Rinderpest virus (RPV).

Key Findings

Mutational Analysis

Residues Gln-37, Tyr-67, Arg-68, Asp-83, Ile-123, and Ser-145 were targeted.

Mutants Q37E, Y67A, R68Q, D83A, and I123A failed to assemble into VLPs, forming partially folded monomers instead.

S145A assembled normally, indicating its non-essential role in folding.

Subunit folding and assembly were shown to be concerted events.

Salt Bridge Studies

D83-R159 salt bridge was dispensable for assembly.

Mutation of Asp-83 disrupted folding, while Arg-159 mutation did not, showing differential roles.

Intermediate Formation

Some double mutants (e.g., D83A-K153Q, R68Q-D150A) formed 18-mer intermediates, representing metastable assembly states.

Epitope Presentation

B-cell epitope from RPV F protein fused at the N-terminus assembled into VLPs but failed to elicit antibodies, likely due to conformational differences or masking by PhMV epitopes.

Fusion into exposed loops or C-terminal regions disrupted folding, yielding insoluble proteins.

CTL epitope fusion at the N-terminus allowed assembly, but biological activity remains to be tested.

Conclusions Subunit folding and assembly in PhMV are tightly coupled processes.

Mutational studies revealed critical residues for folding and assembly.

PhMV can form stable VLPs but may not be an ideal carrier for foreign epitopes, as insertions often disrupt folding.

These findings provide insights into viral assembly mechanisms and highlight limitations of PhMV as a vaccine carrier system.