Submitted to: Journal of Virology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/29/2008
Publication Date: 3/19/2008
Citation: Lim, H-S., Bragg, J.N., Ganesan, U., Lawrence, D.M., Yu, J., Isogai, M., Hammond, J., and Jackson, A.O. 2008. Triple gene block protein interactions involved in movement of Barley stripe mosaic virus. Journal of Virology. 82:4991-5006. Interpretive Summary: An increased understanding of the ways in which virus infection spreads throughout the host plant may lead to new ways of interfering in the infection process and thus conferring protection to plants. Different kinds of plant virus utilize different types of movement proteins to aid in the infection process; Barley stripe mosaic virus (BSMV), which causes an important disease of barley, has a ‘triple gene block’ of proteins that interact to fulfill this function. The interactions between the three ‘triple gene block’ movement proteins of BSMV were studied by a variety of methods, including the introduction of specific mutations into infectious cloned copies of BSMV. Certain mutations disrupted the interactions between the proteins, and critical features of the structure of each of the three ‘triple gene block’ proteins were identified. The ratio of the expression levels of the different gene products was also shown to be critical for efficient virus spread within infected plants. The multiple levels of interaction identified for the BSMV ‘triple gene block’ proteins may lead to increased understanding of the way in which other viruses spread in infected plants, and to new means of conferring virus resistance.
Technical Abstract: The Barley stripe mosaic virus (BSMV) genome contains three movement proteins encoded in an overlapping triple gene block (TGB). The TGB1 (58 kDa), TGB2 (14 kDa), and TGB3 (17 kDa) proteins are each required for cell-to-cell movement of BSMV, and TGB1 binds to ssRNA and dsRNA. We have now isolated a ribonucleoprotein (RNP) complex consisting of TGB1 and plus-sense BSMV genomic (g) and subgenomic (sg) RNAs that may be involved in cell-to-cell movement, and chemical cross-linking of infected barley extracts also revealed high molecular weight TGB1 species that verify TGB1 interactions in planta. Homologous TGB1 binding detected in affinity chromatography assays was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs, but mutations in two C-terminal helicase motifs failed to affect TGB1 interactions. BSMV TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two hybrid experiments demonstrated that TGB1 binds to TGB3, and that TGB2 and TGB3 form heterologous interactions. The latter interactions require the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing substitutions of these amino acids were unable to move from cell to cell. Elevated expression of the wild type TGB3 protein interfered with cell-to-cell movement, but over-expression of a TGB3 mutant that fails to interact with TGB2 did not affect movement. These experiments demonstrate that efficient virus movement requires interactions of TGB2 and TGB3, and also show that substantial deviations from the ratios of the TGB proteins expressed by the wild type virus compromise movement.