Phosphorylation of bamboo mosaic virus satellite RNA (satBaMV)-encoded protein P20 downregulates the formation of satBaMV-P20 ribonucleoprotein complex.

Bamboo mosaic virus (BaMV) satellite RNA (satBaMV) depends on BaMV for its replication and encapsidation. SatBaMV-encoded P20 protein is an RNA-binding protein that facilitates satBaMV systemic movement in co-infected plants. Here, we examined phosphorylation of P20 and its regulatory functions. Recombinant P20 (rP20) was phosphorylated by host cellular kinase(s) in vitro, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and mutational analyses revealed Ser-11 as the phosphorylation site. The phosphor-mimic rP20 protein interactions with satBaMV-translated mutant P20 were affected. In overlay assay, the Asp mutation at S11 (S11D) completely abolished the self-interaction of rP20 and significantly inhibited the interaction with both the WT and S11A rP20. In chemical cross-linking assays, S11D failed to oligomerize. Electrophoretic mobility shift assay and subsequent Hill transformation analysis revealed a low affinity of the phospho-mimicking rP20 for satBaMV RNA. Substantial modulation of satBaMV RNA conformation upon interaction with nonphospho-mimic rP20 in circular dichroism analysis indicated formation of stable satBaMV ribonucleoprotein complexes. The dissimilar satBaMV translation regulation of the nonphospho- and phospho-mimic rP20 suggests that phosphorylation of P20 in the ribonucleoprotein complex converts the translation-incompetent satBaMV RNA to messenger RNA. The phospho-deficient or phospho-mimicking P20 mutant of satBaMV delayed the systemic spread of satBaMV in co-infected Nicotiana benthamiana with BaMV. Thus, satBaMV likely regulates the formation of satBaMV RNP complex during co-infection in planta.

The arginine-rich motif of Bamboo mosaic virus satellite RNA-encoded P20 mediates self-interaction, intracellular targeting, and cell-to-cell movement.

Satellite RNA of Bamboo mosaic virus (satBaMV) has a single open reading frame for a nonstructural, RNA-binding protein, P20, which facilitates the long-distance movement of satBaMV in Nicotiana benthamiana. Here, we elucidate various biological properties of P20 and the involvement of a single domain in its activities. P20 displayed a strong self-interaction in vitro and in vivo, and cross-linking assays demonstrated its oligomerization. Domain mapping, using the bacterial two-hybrid system, indicated that the self-interacting domain overlaps the RNA-binding domain in the N-terminal arginine-rich motif (ARM) of P20. The deletion of the ARM abolished the self-interaction of P20 in vitro and in vivo and impaired its intracellular targeting and efficient cell-to-cell movement in N. benthamiana leaves. Moreover, RNA and protein accumulation of the ARM deletion mutant of satBaMV was significantly reduced in leaves systemically coinfected with Bamboo mosaic potexvirus and satBaMV. This is the first report of the involvement of ARM in various biological activities of a satellite RNA-encoded protein during infection of its host.