Regulation of positive-strand RNA virus replication: The emerging role of phosphorylation.

Protein phosphorylation is a reversible post-translational modification that plays a fundamental role in the regulation of many cellular processes. Phosphorylation can modulate protein properties such as enzymatic activity, stability, subcellular localization or interaction with binding partners. The importance of phosphorylation of the replication proteins of negative-strand RNA viruses has previously been documented but recent evidence suggests that replication of positive-strand RNA viruses - the largest class of viruses, including significant human, animal and plant pathogens - may also be regulated by phosphorylation events. The objective of this review is to summarize current knowledge regarding the various regulatory roles played by phosphorylation of nonstructural viral proteins in the replication of positive-strand RNA viruses

Contribution a l'identificatin des sequences virales impliquees dans la pathogenicite du virus des nervures jaunes et necrotiques de la betterave (BNYVV): apport de la transcription in vitro et de la mutagenese dirigee

SIGLEINIST T 71989 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Etude du complexe de réplication du virus de la mosaïque jaune du navet (TYMV) (assemblage et régulation)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Ubiquitin and plant viruses, let's play together!

International audienc

Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus-insect cell system

UMR BGPI Equipe 2International audienceAll RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome. We have expressed and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST sequence

A viral deubiquitylating enzyme targets viral RNA-dependent RNA polymerase and affects viral infectivity.

International audienceSelective 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

Proteolytic Processing of Turnip Yellow Mosaic Virus Replication Proteins and Functional Impact on Infectivity▿

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus belonging to the alphavirus-like supergroup, encodes its nonstructural replication proteins as a 206K precursor with domains indicative of methyltransferase (MT), proteinase (PRO), NTPase/helicase (HEL), and polymerase (POL) activities. Subsequent processing of 206K generates a 66K protein encompassing the POL domain and uncharacterized 115K and 85K proteins. Here, we demonstrate that TYMV proteinase mediates an additional cleavage between the PRO and HEL domains of the polyprotein, generating the 115K protein and a 42K protein encompassing the HEL domain that can be detected in plant cells using a specific antiserum. Deletion and substitution mutagenesis experiments and sequence comparisons indicate that the scissile bond is located between residues Ser879 and Gln880. The 85K protein is generated by a host proteinase and is likely to result from nonspecific proteolytic degradation occurring during protein sample extraction or analysis. We also report that TYMV proteinase has the ability to process substrates in trans in vivo. Finally, we examined the processing of the 206K protein containing native, mutated, or shuffled cleavage sites and analyzed the effects of cleavage mutations on viral infectivity and RNA synthesis by performing reverse-genetics experiments. We present evidence that PRO/HEL cleavage is critical for productive virus infection and that the impaired infectivity of PRO/HEL cleavage mutants is due mainly to defective synthesis of positive-strand RNA

A Turnip yellow mosaic virus infection system in Arabidopsis suspension cell culture

AbstractTurnip yellow mosaic virus (TYMV) is a positive-strand RNA virus able to infect Arabidopsis thaliana. To establish a TYMV infection system in Arabidopsis cell culture, TYMV replicons with the capsid protein gene replaced by a reporter gene expressing the Sh ble protein conferring zeocin resistance were used to transfect Arabidopsis cells. Zeocin-resistant Arabidopsis calli were used to generate a suspension cell culture. Detection of viral proteins and RNAs after 18 months in culture demonstrated persistent replication of the replicon. The Arabidopsis cell culture yielded soluble, active replication complexes, providing a useful tool to study host factors involved in TYMV replication