Functional Characterization of the Nuclear Localization Signal for a Suppressor of Posttranscriptional Gene Silencing

Abstract

The nucleus-localized C2 protein of Tomato yellow leaf curl virus-China (TYLCV-C) is an active suppressor of posttranscriptional gene silencing (PTGS). Consistently, infection with TYLCV-C resulted in PTGS arrest in plants. The C2 protein possesses a functional, arginine-rich nuclear localization signal within the basic amino acid-rich region 17 KVQHRIAKKTTRRRR 31 . When expressed from potato virus X, C2-RRRR 31 DVGG (in which the four consecutive arginine residues 28 RRRR 31 were replaced with DVGG) that had been tagged with a green fluorescent protein (GFP) failed to transport GFP into nuclei and was dysfunctional in inducing necrosis and suppressing PTGS in plants. Amino acid substitution mutants C2-K 17 D-GFP, C2-HR 21 DV-GFP, and C2-KK 25 DI-GFP localized to nuclei and produced necrosis, but only C2-K 17 D-GFP suppressed PTGS. The N-terminal portions C2 1-31 and C2 17-31 fused in frame to GFP were capable of targeting GFP to nuclei, but neither caused necrosis nor affected PTGS. Our data establish that nuclear localization is likely required for C2 protein to function in C2-mediated induction of necrosis and suppression of PTGS, which may follow diverse pathways in plants. Possible mechanisms of how the C2 protein involves these biological functions are discussed.Posttranscriptional gene silencing (PTGS), RNA interference, and gene quelling represent a conserved cellular defense system for controlling foreign gene expression across the plant, animal, and fungal kingdoms (2,6,8,17,32,42,45). These silencing systems involve double-stranded RNA (dsRNA) from which a 21-to 26-nucleotide (nt) short interfering RNA (siRNA) is derived by the action of an RNase III-like dicer, and they share a common molecular mechanism in which a target RNA is degraded in an RNA homology-dependent manner by an RNA-induced silencing multisubunit RNase complex under the guidance of siRNA (2,3,(15)(16)(17)32). In plants, PTGS defends the host against virus infection, down-regulates transgene expression, and may also participate in the control of development (42,45). To counterattack, plant viruses have evolved the ability to encode proteins (i.e., PTGS suppressors) capable of suppressing PTGS by targeting various stages of the PTGS process, including initiation, propagation, and maintenance (6,29,30,42,45). For example, the potyvirus protein HC-Pro affects PTGS maintenance by interfering with a step coincident with, or upstream of, the production of siRNAs (25,27). HC-Pro interacts with a calmodulin-related protein that can suppress PTGS in plants (1). However, the p25 cell-to-cell movement protein of Potato virus X (PVX) and the 2b protein of Cucumber mosaic virus (CMV) preclude the spread of silencing signals (13, 43). On the other hand, the viral PTGS suppressors are often found to be pathogenicity determinants, and their PTGS suppression activity is associated with pathogenicity determination (44). It is worth noting that certain mutants of the CMV 2b protein have been reported to be functional in pathogenesis but dysfunctional in...

Suppression of RNA Silencing by a Geminivirus Nuclear Protein, AC2, Correlates with Transactivation of Host Genes

“…Studies on AC2 of African cassava mosaic virus (ACMV) and the homologous C2 of Tomato yellow leaf curl virus-China (TYLCCNV), a related monopartite begomovirus, have implicated these proteins in suppression of RNA silencing (10,17,50,53). Notably, TYLCCN C2 requires functional NLS and Zn-finger domains to suppress silencing (10,50). In this work, we found that AC2 from MYMV serves as a transactivator of viral promoter and as a suppressor of RNA silencing.…”

“…The latter suggests that the KKR motif (mutated in NLS1 Ϫ ) is an essential part of the AC2 NLS. However, in the C2 protein of TYLCCNV, the corresponding KKT26DIT mutation did not abolish nuclear localization, in contrast to mutation RRRR31DVGG in the neighboring motif (10). We therefore also mutated the corresponding motif in the MYMV AC2 (RRSR31AASA [NLS2-]).…”

“…Consistent with its function as a transcriptional activator, three conserved domains have been recognized in this protein: a basic domain with a nuclear localization signal (NLS) at the N terminus, a central DNA-binding domain with a nonclassical Zn-finger motif, and an acidic activator domain at the C terminus (21). Studies on AC2 of African cassava mosaic virus (ACMV) and the homologous C2 of Tomato yellow leaf curl virus-China (TYLCCNV), a related monopartite begomovirus, have implicated these proteins in suppression of RNA silencing (10,17,50,53). Notably, TYLCCN C2 requires functional NLS and Zn-finger domains to suppress silencing (10,50).…”

Functional and intracellular localization properties of U6 promoter-expressed siRNAs, shRNAs, and chimeric VA1 shRNAs in mammalian cells

“…In Caenorhabditis elegans, nuclear proteins are reportedly required for RNAi (Dudley et al 2002). It has also been suggested that 21-to 22-nt siRNAs are produced in the nucleus of plants by a nuclear Dicer-like protein (Papp et al 2003), and that PTGS can occur in the nucleus of plants (Dong et al 2003;Papp et al 2003). Additionally, siRNAmediated degradation of the nuclear-localized 7SK RNA (Robb et al 2005) and DMPK mRNA (Langlois et al 2005) has been shown to occur in human cells.…”

Roles of microRNA in plant defense and virus offense interaction

“…In general, viruses accomplish counter-defense by targeting RNAs (Guo and Ding 2002;Thomas et al 2003;Qu et al 2003) or protein (Anandalakshmi et al 2000). Therefore, expression of host genes is modified or silenced (Dong et al 2003). Recent findings suggest that silencing suppressors can contribute to viral symptoms in two ways: helping virus accumulation indirectly and modifying endogenous short-RNA-regulated pathways directly (Silhavy and Burgyán 2004).…”