ИСТИНА

Plant viruses encode movement proteins (MPs) that ensure the viral transport through plasmodesmata (PD) and to neighboring cells. MPs use cell cytoskeleton and endomembranes, mostly the endoplasmic reticulum (ER), for delivery of viral genomes to PD and formation of PD-anchored virus replication compartments. Previously, a novel specialized transport module has been identified in Hibiscus green spot virus (HGSV) and called a ‘binary movement block’ (BMB). BMB codes for two MPs termed BMB1 and BMB2. Using Nicotiana benthamiana transient expression system and confocal laser scanning microscopy. we have found that the integral ER protein BMB2 fused to mRFP is localized to cell wall-embedded punctate structures colocalizing with callose deposits at PDs and in cell wall-adjacent elongated bodies at the cell periphery. Electron microscopy (EM) shows these bodies consist of curved ER tubules. GFP-fused BMB1 is distributed diffusely in the cytoplasm and the nucleus. However, in the presence of BMB2, GFP-BMB1 is found to be directed to cell wall-adjacent bodies, to punctate structures colocalizing with callose deposits at PDs, and to cells adjacent to the initially transformed cell. Thus, BMB2 can direct intracellular trafficking of BMB1 to and through PDs. Further, we demonstrate that the BMB2 induces constrictions of ER tubules, decreases the mobility of ER luminal content as shown by use of fluorescence recovery after photobleaching , and exhibits an affinity to highly curved membranes. These properties are similar to those described for reticulons, cellular proteins that induce membrane curvature to shape the ER tubules. BiFC approach reveals that BMB2, similar to reticulons, adopts a W- like topology within the ER membrane. BMB2 targets PD and increases their size exclusion limit, and these BMB2 activities correlate with the ability to induce constrictions of ER tubules. We propose that the induction of ER constrictions contributes to both the BMB2-dependent increase of the PD permeability and formation of the PD-associated replication compartments, therefore facilitating the virus intercellular spread. Additionally, we show that the ER tubule constrictions also occur in cells expressing TGB2, one of the three MPs of Potato virus X (PVX), and in PVXinfected cells, suggesting that reticulon-like MPs are employed by diverse RNA viruses. The role of cytoskeleton in HGSV cell-to-cell transport is still to be clarified. The actin cytoskeleton has been shown to be involved in subcellular transport BMB2 to PDs by lateral translocation in the ER membrane. Additionally, EM reveals microtubules as one of the major components of cell wall-adjacent BMB2-containing bodies, suggesting that microtubules may take a part in both viral transport and formation of virus replicative compartments. The work was supported by CNRS-RFBR Joint Research Project