Alfalfa mosaic virus replication: Virus factories and host protein interactions

Heather Marie Hutchens, Purdue University

Abstract

Elucidating the mechanisms involved in replication of Alfalfa mosaic virus (AMV) will contribute to both plant pathology and animal virology. Part of this dissertation focuses on determining the location of virus replication in the cell and the mechanism that targets the AMV replication complex. With confocal microscopy of live cells, it was shown that the two AMV replicase proteins, P1 and P2, associate with the tonoplast during infection. When expressed alone, P1-GFP colocalizes with the endosome and multivesicular bodies (MVB). When P1 and P2 are expressed together, P1 recruits P2 to MVB-like structures. With the addition of at least one of the full length AMV RNAs and P2, P1-GFP associates with the tonoplast, suggesting that formation of an active replicase complex is required for transport of the complex to the tonoplast through the prevacuolar compartment (PVC). Because full length RNA is required, it is likely that the 5' and/or 3' UTRs of the AMV RNA play a role in this targeting. Identifying the host proteins that interact with AMV proteins will help define the strategies that AMV employs to evade the cell's defense system to replicate and move to the next cell. Using a yeast two-hybrid assay, AMV coat protein (CP) was shown to interact with PsbP, which is a component of the oxygen evolving complex (OEC) of Photosystem II (PSII). This interaction was confirmed in Nicotiana benthamiana by bimolecular fluorescence complementation (BiFC). BiFC assays of CP mutants indicated that the N-terminus of AMV CP was not required for PsbP and CP interaction; however, the C-terminal 37 amino acids were required, which suggests that this region of CP interacts with PsbP or that CP dimerization, which requires the C-terminal region, is involved in PsbP-CP interaction. To further investigate under what conditions CP and PsbP interact, CP mutants were made with the 191st, 197th and 198th amino acids changed to alanines and the AMV CP and PsbP interaction was disrupted. This indicates that AMV CP dimerization is needed for the interaction with PsbP to occur. To determine whether PsbP positively or negatively affects AMV replication, we investigated the virus susceptibility of N. tabacum plants that have been silenced for PsbP expression by RNAi. It was shown that PsbP is down-regulated during AMV infection, and that the down-regulated plants were more susceptible to AMV infection.

Degree

Ph.D.

Advisors

Loesch-Fries, Purdue University.

Subject Area

Molecular biology|Plant biology|Virology

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