Phosphorylation of viral RNA-dependent RNA polymerase and its role in replication of a plus-strand RNA virus.
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.
Jakubiec
Anna
A
Institut Jacques Monod, 75251 Paris, France.
Tournier
Vincent
V
Institut Jacques Monod, 75251 Paris, France.
Drugeon
Gabrièle
G
Institut Jacques Monod, 75251 Paris, France.
Pflieger
Stéphanie
S
Institut Jacques Monod, 75251 Paris, France.
Camborde
Laurent
L
Institut Jacques Monod, 75251 Paris, France.
Vinh
Joëlle
J
Ecole Supérieure de Physique et Chimie Industrielles de la Ville de Paris, 75005 Paris, France.
Héricourt
François
F
Institut Jacques Monod, 75251 Paris, France.
Redeker
Virginie
V
Ecole Supérieure de Physique et Chimie Industrielles de la Ville de Paris, 75005 Paris, France.
Jupin
Isabelle
I
Institut Jacques Monod, 75251 Paris, France. Electronic address: jupin@ccr.jussieu.fr.
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000378:metabolism / Q000821:virology; D004252:DNA Mutational Analysis; D007313:Insecta; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D012328:RNA Viruses / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D014357:Trypsin / Q000494:pharmacology; D017806:Tymovirus / Q000235:genetics
D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D014357:Trypsin