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

Anna Jakubiec,Vincent Tournier,Gabrièle Drugeon,Stéphanie Pflieger,Laurent Camborde,Joëlle Vinh,François Héricourt,Virginie Redeker,Isabelle Jupin

doi:10.1074/jbc.m600052200

Anna Jakubiec, Vincent Tournier + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m600052200

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Abstract

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.

Highlights

There is growing evidence that phosphorylation of viral proteins plays an important role in regulating viral replication
Turnip yellow mosaic virus (TYMV) 66K Is Phosphorylated in Plant Cells—TYMV 66K RNA-dependent RNA polymerase (RdRp) produced in insect cells is phosphorylated on serine and threonine residues [5]
Coexpression of 140K inhibited the mobility shift of 66K (Fig. 8C, lane 4), while coexpression of the coat protein (CP) as a control had no effect. These results indicate that expression of the 140K replication protein antagonizes 66K phosphorylation at certain sites, in particular Thr64, phosphorylation of which is visible as the 66K mobility shift, and suggest that phosphorylation of the RdRp might be inhibited during viral RNA replication

Summary

The abbreviations used are

RdRp, RNA-dependent RNA polymerase; TYMV, Turnip yellow mosaic virus; CP, coat protein; HPLC, high performance liquid chromatography; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; PSD, post-source decay; nano-ES, nanoelectrospray; hpt, hour post-transfection; EGFP, enhanced green fluorescent protein; RRL, rabbit reticulocyte lysate; WG, wheat germ; WT, wild type. RdRp Phosphorylation and Virus Replication protein encompassing the RdRp domain [10]. Both 140K and 66K are essential for TYMV RNA replication [11], and assembly of TYMV replication complexes, which are located at the periphery of chloroplasts, depends on interactions between the RdRp 66K and the membrane-bound 140K [12, 13]. We demonstrate that TYMV RdRp is phosphorylated in plant cells, when expressed both individually and in the context of viral infection. Systematic mutational analysis of the corresponding residues was used to investigate the correlation between RdRp phosphorylation, assembly of replication complexes, and viral infectivity. The results obtained support the idea that phosphorylationdependent regulatory mechanisms are essential for viral RdRp function and virus replication

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION