Replication of positive-strand RNA viruses, the largest group of plant viruses, is initiated by viral RNA-dependent RNA polymerase (RdRp). Given its essential function in viral replication, understanding the regulation of RdRp is of great importance. Here, we show that Turnip yellow mosaic virus (TYMV) RdRp (termed 66K) is degraded by the proteasome at late time points during viral infection and that the accumulation level of 66K affects viral RNA replication in infected Arabidopsis thaliana cells. We mapped the cis-determinants responsible for 66K degradation within its N-terminal noncatalytic domain, but we conclude that 66K is not a natural N-end rule substrate. Instead, we show that a proposed PEST sequence within 66K functions as a transferable degradation motif. In addition, several Lys residues that constitute target sites for ubiquitylation were mapped; mutation of these Lys residues leads to stabilization of 66K. Altogether, these results demonstrate that TYMV RdRp is a target of the ubiquitin-proteasome system in plant cells and support the idea that proteasomal degradation may constitute yet another fundamental level of regulation of viral replication.

Selective protein degradation via the ubiquitin-proteasome system (UPS) plays an essential role in many major cellular processes, including host-pathogen interactions. We previously reported that the tightly regulated viral RNA-dependent RNA polymerase (RdRp) of the positive-strand RNA virus Turnip yellow mosaic virus (TYMV) is degraded by the UPS in infected cells, a process that affects viral infectivity. Here, we show that the TYMV 98K replication protein can counteract this degradation process thanks to its proteinase domain. In-vitro assays revealed that the recombinant proteinase domain is a functional ovarian tumour (OTU)-like deubiquitylating enzyme (DUB), as is the 98K produced during viral infection. We also demonstrate that 98K mediates in-vivo deubiquitylation of TYMV RdRp protein-its binding partner within replication complexes-leading to its stabilization. Finally, we show that this DUB activity contributes to viral infectivity in plant cells. The identification of viral RdRp as a specific substrate of the viral DUB enzyme thus reveals the intricate interplay between ubiquitylation, deubiquitylation and the interaction between viral proteins in controlling levels of RdRp and viral infectivity.