Phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus adjacent to the RNA binding site affects viral RNA replication

Shapka, Natalia; Stork, Jozsef; Nagy, Peter D.

doi:10.1016/j.virol.2005.08.006

Cited by 40 publications

(33 citation statements)

References 42 publications

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“…Although well documented in negative-strand RNA viruses (39 -41), the role of phosphorylation in regulating the activities and functions of nonstructural proteins in plus-strand RNA viral genomes is only now beginning to emerge (4,6,7,(42)(43)(44)(45). In this study, we present a documented characterization of phosphorylation sites of a viral RdRp.…”

Section: Discussionmentioning

confidence: 93%

Phosphorylation of Viral RNA-dependent RNA Polymerase and Its Role in Replication of a Plus-strand RNA Virus

Jakubiec

Tournier

Drugeon

et al. 2006

Journal of Biological Chemistry

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Central to the process of plus-strand RNA virus genome amplification is the viral RNA-dependent RNA polymerase (RdRp). Understanding its regulation is of great importance given its essential function in viral replication and the common architecture and catalytic mechanism of polymerases. Here we show that Turnip yellow mosaic virus (TYMV) RdRp is phosphorylated, when expressed both individually and in the context of viral infection. Using a comprehensive biochemical approach, including metabolic labeling and mass spectrometry analyses, phosphorylation sites were mapped within an N-terminal PEST sequence and within the highly conserved palm subdomain of RNA polymerases. Systematic mutational analysis of the corresponding residues in a reverse genetic system demonstrated their importance for TYMV infectivity. Upon mutation of the phosphorylation sites, distinct steps of the viral cycle appeared affected, but in contrast to other plus-strand RNA viruses, the interaction between viral replication proteins was unaltered. Our results also highlighted the role of another TYMV-encoded replication protein as an antagonistic protein that may prevent the inhibitory effect of RdRp phosphorylation on viral infectivity. Based on these data, we propose that phosphorylation-dependent regulatory mechanisms are essential for viral RdRp function and virus replication.The reversible phosphorylation of cellular proteins provides a means to induce rapid and profound changes in the function or stability of a protein, and is recognized as a major process by which many aspects of cell biology are regulated. In the field of plus-strand RNA viruses, the largest class of viruses (1) that includes significant pathogens of humans, animals, and plants, there is growing evidence that phosphorylation of viral proteins plays an important role in regulating viral replication.The replication of plus-strand RNA virus genomes depends upon assembly of an intricate replication complex comprising both virus and host components (2). Within this complex, the viral RNA-dependent RNA polymerase (RdRp) 5 plays a pivotal role in the viral infection process, catalyzing synthesis of new viral RNA genomes from the original infecting RNA in a twostage process. The viral genomic plus-strand RNA first acts as a template for the synthesis of a complementary (minus-strand) RNA, which in turn directs synthesis of progeny plus-strand RNA. During replication, a large excess of plus-over minus-

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Section: Discussionmentioning

confidence: 93%

Phosphorylation of Viral RNA-dependent RNA Polymerase and Its Role in Replication of a Plus-strand RNA Virus

Jakubiec

Tournier

Drugeon

et al. 2006

Journal of Biological Chemistry

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“…We showed previously that the bC1 protein binds to DNA in a sequence-nonspecific manner, functions as a suppressor of RNA silencing, and is a pathogenicity protein that plays a vital role in symptom induction by suppression of the silencing defenses in plants (Cui et al, 2005). Phosphorylation of proteins can regulate their nucleic acid-binding properties (Boyle et al, 1991;Mayrand et al, 1993) and interactions between viral RNA and replication proteins of positive-strand RNA viruses (Shapka et al, 2005;Stork et al, 2005). Phosphorylation of bC1 may inhibit its ability to bind nucleic acid, which may negatively impact bC1 function as an RNA-silencing suppressor and result in attenuating viral infection.…”

Section: Discussionmentioning

confidence: 99%

Tomato SlSnRK1 Protein Interacts with and Phosphorylates βC1, a Pathogenesis Protein Encoded by a Geminivirus β-Satellite

et al. 2011

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The bC1 protein of tomato yellow leaf curl China b-satellite functions as a pathogenicity determinant. To better understand the molecular basis of bC1 in pathogenicity, a yeast two-hybrid screen of a tomato (Solanum lycopersicum) cDNA library was carried out using bC1 as bait. bC1 interacted with a tomato SUCROSE-NONFERMENTING1-related kinase designated as SlSnRK1. Their interaction was confirmed using a bimolecular fluorescence complementation assay in Nicotiana benthamiana cells. Plants overexpressing SnRK1 were delayed for symptom appearance and contained lower levels of viral and satellite DNA, while plants silenced for SnRK1 expression developed symptoms earlier and accumulated higher levels of viral DNA. In vitro kinase assays showed that bC1 is phosphorylated by SlSnRK1 mainly on serine at position 33 and threonine at position 78. Plants infected with bC1 mutants containing phosphorylation-mimic aspartate residues in place of serine-33 and/or threonine-78 displayed delayed and attenuated symptoms and accumulated lower levels of viral DNA, while plants infected with phosphorylation-negative alanine mutants contained higher levels of viral DNA. These results suggested that the SlSnRK1 protein attenuates geminivirus infection by interacting with and phosphorylating the bC1 protein.

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“…It is well established that posttranslational modification of proteins is an essential component for many plant regulatory pathways (Shapka et al, 2005;del Pozo et al, 2006;Lindermayr et al, (42) 38 (58) His-Cm-PP16-1 S12A þ F-dextran 20 19 (95) 1 (5) 0 (0) a Cell-to-cell movement of each fluorescent probe was analyzed 2 min after injection into an N. benthamiana mesophyll cell. A Leica confocal laser scanning microscope was used to record the extent of movement; this was categorized as extensive (probe spread out from the injected cell into 10 to 20 neighboring mesophyll cells), limited (probe moved into one neighboring cell), or none (probe remained in injected cell).…”

Section: Phloem Phosphoprotein Compositionmentioning

confidence: 99%

Reciprocal Phosphorylation and Glycosylation Recognition Motifs Control NCAPP1 Interaction with Pumpkin Phloem Proteins and Their Cell-to-Cell Movement

et al. 2007

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In plants, cell-to-cell trafficking of non-cell-autonomous proteins (NCAPs) involves protein-protein interactions, and a role for posttranslational modification has been implicated. In this study, proteins contained in pumpkin (Cucurbita maxima cv Big Max) phloem sap were used as a source of NCAPs to further explore the molecular basis for selective NCAP trafficking. Protein overlay assays and coimmunoprecipitation experiments established that phosphorylation and glycosylation, on both Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (Nt-NCAPP1) and the phloem NCAPs, are essential for their interaction. Detailed molecular analysis of a representative phloem NCAP, Cm-PP16-1, identified the specific residues on which glycosylation and phosphorylation must occur for effective binding to NCAPP1. Microinjection studies confirmed that posttranslational modification on these residues is essential for cell-to-cell movement of Cm-PP16-1. Lastly, a glutathione S-transferase (GST)-Cm-PP16-1 fusion protein system was employed to test whether the peptide region spanning these residues was required for cell-to-cell movement. These studies established that a 36-amino acid peptide was sufficient to impart cell-to-cell movement capacity to GST, a normally cell-autonomous protein. These findings are consistent with the hypothesis that a phosphorylation-glycosylation recognition motif functions to control the binding of a specific subset of phloem NCAPs to NCAPP1 and their subsequent transport through plasmodesmata.

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Phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus adjacent to the RNA binding site affects viral RNA replication

Cited by 40 publications

References 42 publications

Phosphorylation of Viral RNA-dependent RNA Polymerase and Its Role in Replication of a Plus-strand RNA Virus

Phosphorylation of Viral RNA-dependent RNA Polymerase and Its Role in Replication of a Plus-strand RNA Virus

Tomato SlSnRK1 Protein Interacts with and Phosphorylates βC1, a Pathogenesis Protein Encoded by a Geminivirus β-Satellite

Reciprocal Phosphorylation and Glycosylation Recognition Motifs Control NCAPP1 Interaction with Pumpkin Phloem Proteins and Their Cell-to-Cell Movement

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