Studies on the functions of alfalfa mosaic virus coat protein: Assembly and replication

Jiwon Choi, Purdue University

Abstract

Alfalfa mosaic virus (AMV) has a single-stranded positive-sense RNA genome of three RNAs, which are encapsidated along with a subgenomic RNA into bacilliform particles. The shell of the particles is made up of coat protein (CP) dimeric subunits arranged in hexamers along the long particle axis and pentamers at the ends. In vitro, the CP assembles to small T = 1 icosahedral particles in the absence of nucleic acids. The CP is an essential protein because it is required for AMV replication, as well as, particle assembly. It is known that the N-terminus of the CP is required for interaction with the genomic RNAs for both replication and assembly. Crystallographic studies have suggested that the C-terminus is involved in dimer formation; however, the importance of the C-terminus on functions of CP is unclear. In this study, the effects of C-terminal changes on in vitro assembly and replication in protoplasts were investigated. Changes were made in two regions of the C-terminus: the arm region that may be important for stacking interactions in the dimer and the hook region at the very end that may interact with the N-terminus of the dimeric partner. Substitution of alanine at W191 in the arm region interfered with dimerization and assembly, while arginine and valine substitutions at A196 and F197, respectively, did not affect dimerization, but resulted in the assembly of very long rod-shaped particles in vitro. A deletion of 9 amino acids at the end of the hook region had no effect; however, a deletion of 18 amino acids interrupted dimerization. All changes that prevented dimer formation prevented particle assembly. These changes also had significant effects on AMV replication. The CP mutants that did not dimerize were not effective in replication; monomers were about 30% as active as dimers in replication assays in protoplasts. Further studies showed that the oligomeric state of the CP did not affect its expression or accumulation in plants cells. However, CP-RNA interactions were affected. A binding assay suggested that dimer-forming CP mutants had higher affinity for AMV RNA than did mutants unable to dimerize. Results of this study confirmed the role of the C-terminus in dimerization and assembly and suggested that the arm region is the “hinge” that allows alternate dimer conformations, resulting in particles of different morphology. In addition, dimeric CP subunits were found to be important for the replication of AMV.

Degree

Ph.D.

Advisors

Loesch-Fries, Purdue University.

Subject Area

Plant pathology|Microbiology|Molecular biology

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