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.

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