The triple-gene-block protein 3 (TGBp3) of Bamboo mosaic virus (BaMV) is an integral endoplasmic reticulum (ER) membrane protein which is assumed to form a membrane complex to deliver the virus intracellularly. However, the virus entity that is delivered to plasmodesmata (PD) and its association with TGBp3-based complexes are not known. Results from chemical extraction and partial proteolysis of TGBp3 in membrane vesicles revealed that TGBp3 has a right-side-out membrane topology; i.e., TGBp3 has its C-terminal tail exposed to the outer surface of ER. Analyses of the TGBp3-specific immunoprecipitate of Sarkosyl-extracted TGBp3-based complex revealed that TGBp1, TGBp2, TGBp3, capsid protein (CP), replicase and viral RNA are potential constituents of virus movement complex. Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion. This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions. In addition, mutational and confocal microscopy analyses revealed that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. Taken together, our results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membrane complex and recruitment of TGBp1 to the PD by this complex.
The cytokine IL‐17A plays critical roles in the pathogenesis of autoimmune diseases. The frequencies of MAP kinase kinase kinase kinase 3 [also named germinal center kinase–like kinase (GLK)]‐overexpressing T cells are correlated with disease severity of systemic lupus erythematosus (SLE). T‐cell–specific GLK‐transgenic mice develop spontaneous autoimmune responses through IL‐17A. GLK signaling selectively stimulates IL‐17A production in murine T cells through inducing aryl hydrocarbon receptor (AhR)–retinoic acid receptor–related orphan nuclear receptor‐³t (ROR‐³t) complex formation. Here, we investigated whether GLK‐induced AhR–ROR‐³t complex in T cells is a therapeutic target for human SLE. The population of GLK+IL‐17A+ T cells was enhanced in the peripheral blood from patients with SLE compared with that of healthy controls using flow cytometry. The receiver operating characteristic curve analysis showed that increased GLK+IL‐17A+ T‐cell population in peripheral blood reflected an active stage of SLE. In addition, peripheral blood T cells from patients with SLE displayed induction of ROR‐³t phosphorylation and the AhR‐ROR‐³t (and AhR–phosphorylated ROR‐³t) complex. Moreover, we identified a small‐molecule inhibitor, verteporfin, that inhibited GLK kinase activity and AhR–ROR‐³t interaction. The small‐molecule inhibitor verteporfin suppressed the disease severity in autoimmune mouse models and IL‐17A production in T cells from patients with SLE. Collectively, the GLK‐induced AhR‐ROR‐³t (and AhR–phosphorylated ROR‐³t) complex is a therapeutic target for the GLKhighIL‐17Ahigh subpopulation of human patients with SLE.—Chuang, H.‐C, Chen, Y.‐M., Chen, M.‐H., Hung, W.‐T., Yang, H.‐Y., Tseng, Y.‐H., Tan, T.‐H. AhR‐ROR‐³t complex is a therapeutic target for MAP4K3/GLKhighIL‐17Ahigh subpopulation of systemic lupus erythematosus. FA8EB J. 33, 11469–11480 (2019). http://www.fasebj.org
The triple gene block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) has been proposed to be a transmembrane protein; however, its features remain unclear. Here, we used biochemical approaches to determine its topological properties. Our data reveal that TGBp2 is mainly associated with the endoplasmic reticulum membrane. The resistance of TGBp2 in proteoliposomes, prepared from both the BaMV-infected tissues and in vitro reconstitution system, to both chemical extraction and trypsin digestion confirmed that it is indeed an integral membrane protein. On the basis of the minor change in the size of the major stable TGBp2-derived tryptic fragment from the monomeric TGBp2, as well as the sensitivity of the cysteine residues at the C-terminal tail of TGBp2 to maleimide modification, we suggest that TGBp2 adopts a topology with both its short N- and C-terminal tails exposed to the outer surface of the endoplasmic reticulum. Moreover, TGBp2 is able to self-assemble as revealed by the significant increase in multimeric TGBp2 when the TGBp2-containing proteoliposomes were treated with chemical crosslinker or oxidation agent.
The triple gene block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) is a transmembrane protein which is known to be required for the cell-to-cell movement of potexviruses. This protein has two conserved Cys residues, Cys-109 and Cys-112, at its C-terminal tail, which is supposed to be exposed on the outer surface of the endoplasmic reticulum (ER) membrane and ER-derived granular vesicles. In this study, we investigated the importance of these two Cys residues on the cell-to-cell and systemic movement of BaMV. Our results indicate that the Cys-to-Ala substitutions in TGBp2 make the cell-to-cell movement of BaMV relatively inefficient and the systemic movement of BaMV severely inhibited. Moreover, the defect in systemic movement is attributed to the inefficient transport of viral RNA in the phloem of petiole. Clearly, TGBp2 is critical not only for the cell-to-cell but also for the systemic movement of BaMV. In addition, the conserved Cys residues are important for the functioning of TGBp2.
The triple-gene-block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) is a transmembrane protein which was proposed to be involved in viral RNA binding during virus transport. Here, we report on the RNA-binding properties of TGBp2. Using tyrosine fluorescence spectroscopy and UV-crosslinking assays, the TGBp2 solubilized with Triton X-100 was found to interact with viral RNA in a non-specific manner. These results raise the possibility that TGBp2 facilitates intracellular delivery of viral RNA through non-specific protein-RNA interaction. FindingsBamboo mosaic virus (BaMV) is a single-stranded, positivesense RNA virus. Its genomic RNA has three partially overlapping open reading frames, called triple gene block (TGB), located between the coding sequences for the replicase and capsid protein [1]. The TGB-encoded proteins are referred to as TGBp1, TGBp2 and TGBp3 according to their positions [2] and are required for virus movement in the host plant [3][4][5][6]. The TGB proteins are found in several different viral genera. On the basis of amino acid sequence comparisons of the TGB proteins, the TGB-containing viruses have been classified into hordei-like and potex-like viruses [7]. Bamboo mosaic virus is a potex-like virus.The functions of each TGB protein have been investigated. TGBp2 is an integral membrane protein with two transmembrane helices [8] and a topology with both its N-and C-terminal tails exposed to the outer surface of endoplasmic reticulum (ER) and the central loop in the lumen of ER [9,10]. Inhibition of virus movement by mutations disrupting the transmembrane helices of Potato virus X (PVX) TGBp2 indicated that ER association is important for the functioning of TGBp2 (8). Moreover, the PVX TGBp2 is able to induce the formation of granular vesicles derived from the ER, which align on actin filaments [11]. Mutations in the central loop region of PVX TGBp2 eliminate the formation of granular vesicles and inhibit the cell-to-cell movement of virus [12]. In addition, the PVX TGBp2 is able to increase the size exclusion limit of plasmodesmata (PD) [13], probably through its association with host interacting proteins (TIPs) which in accompany with β-1, 3-glucanase regulate callose degradation [14].The membrane-associated TGBp2 is thought to assist the intracellular transport of the viral ribonucleoprotein
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