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

Abstract

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 Nucleolar Fibrillarin Protein Is Required for Helper Virus-Independent Long-Distance Trafficking of a Subviral Satellite RNA in Plants

“…The P20 Protein Plays a Crucial Role in SatBaMV Systemic Movement in N. benthamiana Previously, we showed that the trafficking of P20-defective satBaMV exhibits low efficiency in N. benthamiana coinfected with BaMV HV (Lin et al, 1996;Vijaya Palani et al, 2006;Vijayapalani et al, 2012). To provide further insights into the role of P20 in the systemic movement of satBaMV, we generated transgenic N. benthamiana expressing the P20 protein under the phloem companion cell-specific SUC2 promoter ( Figure 1A).…”

“…Unlike most satRNA-encoded proteins required for satRNA replication (Hu et al, 2009), the satBaMV-encoded P20 nonstructural protein assists satBaMV long-distance trafficking in plants (Lin et al, 1996;Vijaya Palani et al, 2006;Vijayapalani et al, 2012) (Figure 1). Previously, we showed that P20 can preferentially bind to the 59-and 39-untranslated regions of satBaMV to form satBaMV-P20 RNP complexes (Tsai et al, 1999;Vijayapalani et al, 2012) and that formation of the complexes is negatively regulated by P20 phosphorylation (Vijayapalani et al, 2012).…”

“…Previously, we showed that P20 can preferentially bind to the 59-and 39-untranslated regions of satBaMV to form satBaMV-P20 RNP complexes (Tsai et al, 1999;Vijayapalani et al, 2012) and that formation of the complexes is negatively regulated by P20 phosphorylation (Vijayapalani et al, 2012). Our current results show that P20 can form punctate structures localized at PD (Figure 7) and the satBaMV-P20 RNP complexes can traffic autonomously through the phloem in satBaMV-transgenic stocks or scions ( Figures 2B to 2D).…”

“…SatBaMV encodes a 20-kD nonstructural RNA binding protein (P20) that is dispensable for replication (Lin et al, 1996) but is required for long-distance satBaMV transport in HV coinfected Nicotiana benthamiana (Vijaya Palani et al, 2006;Vijayapalani et al, 2012). The P20 protein has several MP features, including RNA binding activity (Tsai et al, 1999), strong self-interactions, and efficient cellto-cell movement (Vijaya Palani et al, 2006).…”

“…The P20 protein has several MP features, including RNA binding activity (Tsai et al, 1999), strong self-interactions, and efficient cellto-cell movement (Vijaya Palani et al, 2006). P20 accumulates in the cytoplasm and nuclei of HV and satBaMV coinfected cells (Palani et al, 2009) and can be phosphorylated to negatively regulate the formation of satBaMV RNP complexes (Vijayapalani et al, 2012). BaMV HV is a member of the Potexvirus genus that contains a single-stranded, positive-sense RNA genome with five open reading frames.…”