No abstract

Several RNA virus genera belonging to the Virgaviridae and Flexiviridae families encode proteins organized in a triple gene block (TGB) that facilitate cell-to-cell and long-distance movement. The TGB proteins have been traditionally classified as hordei-like or potex-like based on phylogenetic comparisons and differences in movement mechanisms of the Hordeivirus and Potexvirus spp. However, accumulating data from other model viruses suggests that a revised framework is needed to accommodate the profound differences in protein interactions occurring during infection and ancillary capsid protein requirements for movement. The goal of this article is to highlight common features of the TGB proteins and salient differences in movement properties exhibited by individual viruses encoding these proteins. We discuss common and divergent aspects of the TGB transport machinery, describe putative nucleoprotein movement complexes, highlight recent data on TGB protein interactions and topological properties, and review membrane associations occurring during subcellular targeting and cell-to-cell movement. We conclude that the existing models cannot be used to explain all TGB viruses, and we propose provisional Potexvirus, Hordeivirus, and Pomovirus models. We also suggest areas that might profit from future research on viruses harboring this intriguing arrangement of movement proteins.

We studied subcellular distribution of green fluorescent protein (GFP)-tagged movement proteins encoded by the second and the third genes of poa semilatent hordeivirus (PSLV) triple gene block (TGB), 15K TGBp2 and 18K TGBp3. GFP-15K transiently expressed in Nicotiana benthamiana leaf epidermal cells was associated with the endomembrane system elements. GFP-18K appeared in the membrane bodies at cell periphery. Mutation analysis demonstrated that subcellular targeting of GFP-15K depended on the protein transmembrane segment(s), whereas the TGBp3 central hydrophilic region was responsible for targeting of GFP-18K. Coexpression of GFP-15K with the intact 18K protein induced drastic changes in the TGBp2 localization: GFP-15K appeared in the cell peripheral bodies similar to those in the cells expressing GFP-18K alone. Coexpression experiments with mutant forms of both proteins argue against involvement of direct interaction between small TGB proteins in the TGBp3-assisted targeting of TGBp2 to the cell peripheral compartments. This conclusion was further confirmed by similar effects on the PSLV 15K TGBp2 localization induced by TGBp3 proteins of PSLV and potato virus X, which have no detectable sequence similarity to each other.

RNA silencing is a natural defense mechanism against genetic stress factors, including viruses. A mutant hordeivirus (Barley stripe mosaic virus [BSMV]) lacking the ␥b gene was confined to inoculated leaves in Nicotiana benthamiana, but systemic infection was observed in transgenic N. benthamiana expressing the potyviral silencing suppressor protein HCpro, suggesting that the ␥b protein may be a long-distance movement factor and have antisilencing activity. This was shown for ␥b proteins of both BSMV and Poa semilatent virus (PSLV), a related hordeivirus. Besides the functions in RNA silencing suppression, ␥b and HCpro had analogous effects on symptoms induced by the hordeiviruses. Severe BSMV-induced symptoms were correlated with high HCpro concentrations in the HCpro-transgenic plants, and substitution of the ␥b cistron of BSMV with that of PSLV led to greatly increased symptom severity and an altered pattern of viral gene expression. The efficient systemic infection with the chimera was followed by the development of dark green islands (localized recovery from infection) in leaves and exemption of new developing leaves from infection. Recovery and the accumulation of short RNAs diagnostic of RNA silencing in the recovered tissues in wild-type N. benthamiana were suppressed in HCpro-transgenic plants. These results provide evidence that potyviral HCpro and hordeivirus ␥b proteins contribute to systemic viral infection, symptom severity, and RNA silencing suppression. HCpro's ability to suppress the recovery of plants from viral infection emphasizes recovery as a manifestation of RNA silencing.The first observations of posttranscriptional gene silencing, or RNA silencing, were made more than 10 years ago. At that time, the phenomenon was described as "cosuppression" of endogenous genes caused by expression of homologous sequences in transgenic plants (43,66). RNA silencing is no longer associated only with transgenic plants but is known as a natural defense system against genetic stress factors, such as viruses and transposable elements, in multicellular eukaryotic organisms (5,6,11,20,35,39,68,70,73). RNA silencing is characterized by rapid and specific degradation of cytoplasmic RNAs. The accumulation of small 21-to 25-nucleotide RNA fragments (small interfering RNAs [siRNAs]) originating from the target sequence is diagnostic of RNA silencing (24, 37). The most potent inducer of RNA silencing is double-stranded RNA (dsRNA) (5,6,11,20,35,39,63,68,70,73). The onset of RNA silencing is followed by a propagation phase during which a systemic signal is delivered from the tissues undergoing silencing and is transported to other parts of the plant where homologous RNA molecules will be silenced. The nature of the signal is yet unknown, but the signaling pathway follows the transport of macromolecules and viruses through plasmodesmata between cells and via phloem over long distances (20,53,64).

TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the "triple gene block" (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus) movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBpl to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.