Triple Gene Block Protein Interactions Involved in Movement of Barley Stripe Mosaic Virus

Abstract

Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The 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. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.For a virus to successfully invade a plant and cause disease, it must have the ability to move from cell to cell, establish localized infection foci, enter and exit the vascular system, and develop systemic infections. To accomplish these activities, plant viruses encode one or more movement proteins (MPs) that facilitate cell-to-cell movement and vascular transport. These proteins generally localize at plasmodesmata (PD) and increase the permeability of the PD sufficiently to permit the movement of macromolecules through the desmotubule (25, 38). Many MPs have RNA binding activities, and some act in concert with other virus-encoded proteins to facilitate virus movement and other activities such as RNA unwinding (18) or suppression of gene silencing (1, 24). Several general classes of viral MPs are known to exist, and these proteins provide tools for investigating a wide range of host-virus interactions and cellular functions (25,26).The most extensively investigated MPs are members of the 30K superfamily that are encoded by a large number of RNA and DNA viruses with different genome organizations (27). Over the past 15 years, studies of the processes carried out by proteins of the 30K movement family have provided great insight into the requirements for local and long-distance transport of Tobacco mosaic virus and a number of other viruses (4,15,25,38). The triple gene block (TGB) superfamily represents anoth...

Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to-cell movement and eliminates long-distance virus movement

“…In the hordeivirus barley stripe mosaic virus (BSMV), TGB3 is produced in approximately one-tenth of the amount of TGB2 (Donald et al, 1993). However, whilst excessive amounts of BSMV or other hordeivirus-like TGB3s interfere with virus replication (Lauber et al, 2005;Lim et al, 2008), there seems to be no such limitation with potexvirus-like TGB3s (Lin et al, 2006; this study). The ability to overexpress TGB3 mutants in cis to complement a TGB3-defective AltMV should allow further examination of the functions of TGB3 for both local and long-distance movement, and of the differences between AltMV and PVX.…”

Domain organization of the N-terminal portion of hordeivirus movement protein TGBp1

“…Although TGBp1-formed RNP complexes isolated from BSMV-infected plants contain only virus-specific ssRNAs of positive polarity (Lim et al, 2008), BSMV TGBp1 also appears to bind dsRNA in vitro (Donald et al, 1997). Moreover, both BSMV and PSLV TGBp1 are able to unwind RNA duplexes in vitro, thus demonstrating the RNA helicase activity (Kalinina et al, 2002).…”

“…TGBp2 and TGBp3 are small membrane proteins necessary for intracellular transport of complexes containing TGBp1 and viral RNA to plasmodesmata (Morozov & Solovyev, 2003;Zamyatnin et al, 2004; Haupt et al, 2005;Jackson et al, 2009 Lim et al, 2008;Jackson et al, 2009). Such TGBp1-RNA complexes are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in plants (Morozov & Solovyev, 2003;Lim et al, 2008). In potex-like TGBs, the whole TGBp1 sequence is represented by the NTPase/helicase domain (HELD) with seven conserved motifs of the superfamily 1 NTPases/helicases (Gorbalenya et al, 1989), whereas the hordei-like TGBp1 has an additional N-terminal 'extension region' (Morozov & Solovyev, 2003).…”

“…The TGB is a conserved module of overlapping genes found in a number of virus groups (Morozov & Solovyev, 2003). Hordeivirus TGBp1 binds viral genomic RNA forming a cell-to-cell transportcompetent complex (Brakke et al, 1988;Kalinina et al, 2001;Lim et al, 2008;Jackson et al, 2009). TGBp2 and TGBp3 are small membrane proteins necessary for intracellular transport of complexes containing TGBp1 and viral RNA to plasmodesmata (Morozov & Solovyev, 2003;Zamyatnin et al, 2004; Haupt et al, 2005;Jackson et al, 2009 Lim et al, 2008;Jackson et al, 2009).…”

“…It should also be kept in mind that the TGBp1 NTPase/helicase domain has its own RNA-binding activity. Therefore, RNP formation by TGBp1 can involve RNA interaction with three different protein domains.Although TGBp1-formed RNP complexes isolated from BSMV-infected plants contain only virus-specific ssRNAs of positive polarity (Lim et al, 2008), BSMV TGBp1 also appears to bind dsRNA in vitro (Donald et al, 1997). Moreover, both BSMV and PSLV TGBp1 are able to unwind RNA duplexes in vitro, thus demonstrating the RNA helicase activity (Kalinina et al, 2002).…”