Abstract
The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMPα]) is predicted to be involved in 2b’s nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b’s C-terminal half as an NES. We then showed that NES-deficient 2b accumulated abundantly in the nucleus and lost its RSS activity, suggesting that 2b exported from the nucleus can play a role as an RSS. Although two serine residues (S40 and S42) were previously found to be phosphorylated, we also found that an additional phosphorylation site (S28) alone can affect 2b’s nuclear localization and RSS activity. Alanine substitution at S28 impaired the IMPα-mediated nuclear/nucleolar localization of 2b, and RSS activity was even stronger compared to wild-type 2b. In a subcellular fractionation assay, phosphorylated 2bs were detected in the nucleus, and comparison of the accumulation levels of nuclear phospho-2b between wild-type 2b and the NES mutant showed a greatly reduced level of the phosphorylated NES mutant in the nucleus, suggesting that 2bs are dephosphorylated in the nucleus and may be translocated to the cytoplasm in a nonphosphorylated form. These results suggest that 2b manipulates its nucleocytoplasmic transport as if it tracks down its targets, small RNAs and AGOs, in the RNA silencing pathway. We infer that 2b’s efficient RSS activity is maintained by a balance of phosphorylation and dephosphorylation, which are coupled to importin/exportin-mediated shuttling between the nucleus and cytoplasm.
Highlights
The nuclear envelope is a physical barrier that regulates dynamic compartmentation of proteins between the nucleus and cytoplasm [1]
The nuclear export of the P6 protein of a DNA virus, cauliflower mosaic virus (CaMV), is mediated by the leucine-rich motif in the Nterminus, which is recognized by an exportin, CRM1 known as XPO1 [14]
Because the serine residue 28 (S28) residue is just adjacent to the two nuclear localization signals (NLSs), and the results of our subcellular localization study indicated that phosphorylation of S28 is required for 2b’s nuclear localization, we considered that phosphorylation of S28 was important for the 2b–IMPα interaction
Summary
The nuclear envelope is a physical barrier that regulates dynamic compartmentation of proteins between the nucleus and cytoplasm [1]. Because this compartmentation of proteins is critical for signal transduction and gene regulation in the context of cell proliferation, differentiation and transformation, nucleocytoplasmic partitioning is accomplished by highly selective processes [2]. The export of a protein from the nucleus is generally mediated by nuclear export signals (NESs). The nuclear export of the P6 protein of a DNA virus, cauliflower mosaic virus (CaMV), is mediated by the leucine-rich motif in the Nterminus, which is recognized by an exportin, CRM1 known as XPO1 [14]. Nucleocytoplasmic shuttling of the tomato leaf carl Yunnan geminivirus (TLCYnV) C4 protein is mediated by post-translational phosphorylation and myristoylation, and its interaction with XPO1 is required for its nuclear export [16]. For RNA viruses, the NES motif of beet necrotic yellow vein virus (BNYVV) protein p25 is involved in p25’s nuclear export [17]
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