Alfalfa mosaic virus: Characterization of coat protein and movement protein
Alfalfa mosaic virus (AMV) is a virus with a single-stranded, plus-sense RNA genome consisting of three RNAs, RNAs 1, 2, and 3. RNAs 1 & 2 encode the replicase proteins, which are involved in AMV RNA synthesis and RNA 3 encodes P3, involved in the movement of virus throughout infected plants, and coat protein (CP) translated only from subgenomic RNA 4. ^ Previous studies of AMV CP showed that the N-terminus of CP is required for interaction with RNA and for the initiation of replication and that the C-terminus of CP was required for dimer formation. To understand the interaction between CP and RNA at the molecular level, N- and C-terminal mutants of the CP were investigated. It was found that AMV CP must have Arg at amino acid position 17 to bind to RNA and to initiate replication. CP mutants with either a Lys substitution at position 17 or Lys16 and Arg17 switched were not active. Amino acids in the C-terminus were also found to be important for these functions as well. However, the primary effect of the C-terminal mutations was on the structure of the CP rather than directly on RNA binding. CP monomers do not bind RNA nor initiate infection as well as dimers. Thus, the activity of all AMY CP mutants in replication correlated with their affinity for AMV RNA. ^ AMV P3 has been shown to have properties shared by other virus movement proteins. To isolate P3 for additional biochemical studies, the protein was over expressed in Pichia pastoris, a methylotrophic yeast, as the native protein (P3) and as a fusion protein with poly (His)6 (P3-F). Both proteins accumulated in a soluble form in the yeast and could be partially purified using a metal-chelated resin or size exclusion chromatography. In vivo labeling and SDS-PAGE of P3 and P3-F suggested that a portion of these proteins was post-translationally modified by phosphorylation. ^
Major Professor: L. Sue Loesch-Fries, Purdue University.
Biology, Molecular|Biology, Microbiology|Agriculture, Plant Pathology
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