Nuclear-Cytoplasmic Partitioning of Cucumber Mosaic Virus Protein 2b Determines the Balance between Its Roles as a Virulence Determinant and an RNA-Silencing Suppressor

Abstract

The Cucumber Mosaic Virus (CMV) 2b protein is an RNA-silencing suppressor that plays roles in CMV accumulation and virulence. The 2b proteins of subgroup IA CMV strains partition between the nucleus and cytoplasm, but the biological significance of this is uncertain. We fused an additional nuclear localization signal (NLS) to the 2b protein of subgroup IA strain Fny-CMV to create 2b-NLS and tested its effects on subcellular distribution, silencing, and virulence. The additional NLS enhanced 2b protein nuclear and nucleolar accumulation, but nuclear and nucleolar enrichment correlated with markedly diminished silencing suppressor activity in patch assays and abolished 2b protein-mediated disruption of microRNA activity in transgenic Arabidopsis. Nucleus/nucleolus-localized 2b protein possesses at least some ability to inhibit antiviral silencing, but this was not sufficient to prevent recovery from disease in younger, developing leaves in Arabidopsis. However, enhanced nuclear and nucleolar accumulation of 2b increased virulence and accelerated symptom appearance in older leaves. Experiments with Arabidopsis lines carrying mutant Dicer-like alleles demonstrated that compromised suppressor activity explained the diminished ability of 2b-NLS to enhance virus accumulation. Remarkably, the increased virulence that 2b-NLS engendered was unrelated to effects on microRNA- or short interfering RNA-regulated host functions. Thus, although nucleus- and nucleolus-localized 2b protein is less efficient at silencing suppression than cytoplasm-localized 2b, it enhances CMV virulence. We propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.IMPORTANCE In this work, the main finding is that nucleus/nucleolus-localized 2b protein is strongly associated with CMV virulence, which is independent of its effect on small RNA pathways. Moreover, this work supports the contention that the silencing suppressor activity of CMV 2b protein is predominantly exerted by that portion of the 2b protein residing in the cytoplasm. Thus, we propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.

Two amino acids near the N‐terminus of Cucumber mosaic virus 2b play critical roles in the suppression of RNA silencing and viral infectivity

“…In spite of the multiple functions of 2b, only dsRNA binding, but not nuclear targeting or interaction with AGOs, is required for suppression of post-transcriptional gene silencing (Duan et al, 2012;González et al, 2010González et al, , 2012. In the context of viral pathogenicity, however, 2b nuclear localization is indispensable for virulence and inhibition of RNA-directed DNA methylation (RdDM) (Du et al, 2014;Duan et al, 2012).…”

N‐terminal region of cysteine‐rich protein (CRP) in carlaviruses is involved in the determination of symptom types

“…In our preliminary observations, PVM CRP localized not only to the nucleus but also to the cytoplasm in leaf epidermal cells of N. benthamiana plants (data not shown). As reported in the CMV 2b protein, which partitions between the nucleus and cytoplasm (Du et al ., ), PVM CRP is probably a nucleus‐ and cytoplasm‐localized protein. CVB CRP was observed mostly in the nucleus, but also in the cytoplasm (Lukhovitskaya et al ., ).…”

“…RSV p3 and CTV p23 are RSSs whose activity requires NLS (Ruiz‐Ruiz et al ., ; Xiong et al ., ). Carlavirus CRPs tested in this study have parallels with BNYVV and BSBMV CRP and CMV 2b protein, in that NLS plays a role in the enhancement of virus pathogenicity, but not in RSS activity (Chiba et al ., ; Du et al ., ; González et al ., ). As carlaviruses are positive‐stranded RNA viruses that replicate in the cytoplasm, most of their encoded proteins, including CRP, do not function in the nucleus in principle.…”

“…As carlaviruses are positive-stranded RNA viruses that replicate in the cytoplasm, most of their encoded proteins, including CRP, do not function in the nucleus in principle. However, it has been reported that the accumulation of 2b of CMV, which is also a positive-stranded RNA virus, in the nucleus enhances virus virulence (Du et al, 2014). In addition, CMV 2b has been shown to be a transcriptional activator in yeast (Ham et al, 1999).…”

Expression of Cucumber mosaic virus suppressor 2b alters FWA methylation and its siRNA accumulation in Arabidopsis thaliana

“…CMV CP and 2b exist in cytoplasmic as well as nuclear fractions (Fig. 4B), signifying their dual roles in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) (Du et al, 2014). Moreover AGO4 redistribution in cytoplasm and its shuttling back to the nucleus along with hcsiRNAs describe its role in hijacking the cytoplasmic hcsiRNAs for methylation in nucleus (Ye et al, 2012).…”